Cell adhesion inhibitors

ABSTRACT

The present invention relates to novel compounds that are useful for inhibition and prevention of cell adhesion and cell adhesion-mediated pathologies. This invention also relates to pharmaceutical formulations comprising these compounds and methods of using them for inhibition and prevention of cell adhesion and cell adhesion-mediated pathologies. The compounds and pharmaceutical compositions of this invention can be used as therapeutic or prophylactic agents. They are particularly well-suited for treatment of many inflammatory and autoimmune diseases.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional (and claims the benefit of priorityunder 35 USC §120) of U.S. application Ser. No. 08/875,321, filed Sep.19, 1997, now U.S. Pat. No. 6,376,538 which is a 371 of PCT/US96/01349,filed Jan. 18, 1996, which is a Continuation-in-Part of U.S.application, Ser. No. 08/376,372, filed Jan. 23, 1995 now U.S. Pat. No.6,306,840.

TECHNICAL FIELD OF THE INVENTION

The present invention relates to novel compounds that are useful forinhibition and prevention of cell adhesion and cell adhesion-mediatedpathologies. This invention also relates to pharmaceutical formulationscomprising these compounds and methods of using them for inhibition andprevention of cell adhesion and cell adhesion-mediated pathologies. Thecompounds and pharmaceutical compositions of this invention can be usedas therapeutic or prophylactic agents. They are particularly well-suitedfor treatment of many inflammatory and autoimmune diseases.

BACKGROUND OF THE INVENTION

Cell adhesion is a process by which cells associate with each other,migrate towards a specific target or localize within the extra-cellularmatrix. As such, cell adhesion constitutes one of the fundamentalmechanisms underlying numerous biological phenomena. For example, celladhesion is responsible for the adhesion of hemoatopoietic cells toendothelial cells and the subsequent migration of those hemopoieticcells out of blood vessels and to the site of injury. As such, celladhesion plays a role in pathologies such as inflammation and immunereactions in mammals.

Investigations into the molecular basis for cell adhesion have revealedthat various cell-surface macromolecules—collectively known as celladhesion molecules or receptors—mediate cell-cell and cell-matrixinteractions. For example, proteins of the superfamily called“integrins” are the key mediators in adhesive interactions betweenhematopoietic cells and their microenvironment (M. E. Hemler, “VLAProteins in the Integrin Family: Structures, Functions, and Their Roleon Leukocytes.”, Ann. Rev. Immunol., 8, p. 365 (1990)). Integrins arenon-covalent heterodimeric complexes consisting of two subunits called αand β. There are at least 12 different α subunits (α1-α6, α-L, α-M, α-X,α-IIB, α-V and α-E) and at least 9 different β (β1-β9) subunits. Basedon the type of its α and β subunit components, each integrin molecule iscategorized into a subfamily.

α4β1 integrin, also known as very late antigen-4 (“VLA-4”), CD49d/CD29,is a leukocyte cell surface receptor that participates in a wide varietyof both cell-cell and cell-matrix adhesive interactions (M. E. Hemler,Ann. Rev. Immunol., 8, p. 365 (1990)). It serves as a receptor for thecytokine-inducible endothelial cell surface protein, vascular celladhesion molecule-1 (“VCAM-1”), as well as to the extracellular matrixprotein fibronectin (“FN”) (Ruegg et al., J. Cell Biol., 177, p. 179(1991); Wayner et al., J. Cell Biol., 105, p. 1873 (1987); Kramer etal., J. Biol. Chem., 264, p. 4684 (1989); Gehlsen et al. Science, 24, p.1228 (1988)). Anti-VLA4 monoclonal antibodies (“mAb's”) have been shownto inhibit VLA4-dependent adhesive interactions both in vitro and invivo (Ferguson et al. Proc. Natl. Acad. Sci., 88, p. 8072 (1991);Ferguson et al., J. Immunol., 150, p. 1172 (1993)). Results of in vivoexperiments suggest that this inhibition of VLA-4-dependent celladhesion may prevent or inhibit several inflammatory and autoimmunepathologies (R. L. Lobb et al., “The Pathophysiologic Role of α-4Integrins In Vivo”, J. Clin. Invest., 94, pp. 1722-28 (1994)).

In order to identify the minimum active amino acid sequence necessary tobind VLA-4, Komoriya et al. (“The Minimal Essential Sequence for a MajorCell Type-Specific Adhesion Site (CS1) Within the Alternatively SplicedType III Connecting Segment Domain of Fibronectin Is Leucine-AsparticAcid-Valine”, J. Biol. Chem., 266 (23), pp. 15075-79 (1991)) synthesizeda variety of overlapping peptides based on the amino acid sequence ofthe CS-1 region (the VLA-4 binding domain) of a particular species offibronectin. They identified an 8-amino acid peptide,Glu-Ile-Leu-Asp-Val-Pro-Ser-Thr [SEQ ID NO: 1], as well as two smalleroverlapping pentapeptides, Glu-Ile-Leu-Asp-Val [SEQ ID NO: 2] andLeu-Asp-Val-Pro-Ser [SEQ ID NO: 3], that possessed inhibitory activityagainst FN-dependent cell adhesion. These results suggested thetripeptide Leu-Asp-Val as a minimum sequence for cell-adhesion activity.It was later shown that Leu-Asp-Val binds only to lymphocytes thatexpress an actived form of VLA-4, thus bringing into question theutility of such a peptide in vivo (E. A. Wayner et al.,“Activation-Dependent Recognition by Hematopoietic Cells of the LDVSequence in the V Region of Fibronectin”, J. Cell. Biol., 116(2), pp.489-497 (1992)). However, certain larger peptides containing the LDVsequence were subsequently shown to be active in vivo [T. A. Ferguson etal., “Two Integrin Binding Peptides Abrogate T-cell-Mediated ImmuneResponses In Vivo,” Proc. Natl. Acad. Sci. USA, 88, pp. 8072-76 (1991);and S. M. Wahl et al., “Synthetic Fibronectin Peptides SuppressArthritis in Rats by Interrupting Leukocyte Adhesion and Recruitment,”J. Clin. Invest., 94, pp. 655-62 (1994)].

A cyclic pentapeptide, Arg-Cys-Asp-TPro-Cys (wherein TPro denotes4-thioproline) (SEQ ID NO:5), which can inhibit both VLA-4 and VLA-5adhesion to FN has also been described (D. M. Nowlin et al. “A NovelCyclic Pentapeptide Inhibits α4β1 and α5 β1 Integrin-mediated CellAdhesion”, J. Biol. Chem., 268(27), pp. 20352-59 (1993); and PCTpublication PCT/US91/04862). This peptide was based on the tripeptuidesequence Arg-Gly-Asp from FN which had been known as a common motif inthe recognition site for several extracellular-matrix proteins.

Despite these advances, there remains a need for small, specificinhibitors of VLA-4-dependent cell adhesion. Ideally, such inhibitorswould be semi-peptidic or non-peptidic so that they may be orallyadministered. Such compounds would provide useful agents for treatment,prevention or suppression of various pathologies mediated by celladhesion and VLA-4 binding.

SUMMARY OF THE INVENTION

The present invention solves this problem by providing novelnon-peptidic compounds that specifically inhibit the binding of ligandsto VLA-4. These compounds are useful for inhibition, prevention andsuppression of VLA-4-mediated cell adhesion and pathologies associatedwith that adhesion, such as inflammation and immune reactions. Thecompounds of this invention may be used alone or in combination withother therapeutic or prophylactic agents to inhibit, prevent or suppresscell adhesion. This invention also provides pharmaceutical formulationscontaining these VLA-4-mediated cell adhesion inhibitors and methods ofusing the compounds and compositions of the invention for inhibition ofcell adhesion.

According to one embodiment of this invention, these novel compounds,compositions and methods are advantageously used to treat inflammatoryand immune diseases. The present invention also provides methods forpreparing the compounds of this invention and intermediates useful inthose methods.

DETAILED DESCRIPTION OF THE INVENTION

Definitions

As used herein, the term “alkyl”, alone or in combination, refers to astraight-chain or branched-chain alkyl radical containing from 1 to 10,preferably from 1 to 6 and more preferably from 1 to 4, carbon atoms.Examples of such radicals include, but are not limited to, methyl,ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl,pentyl, iso-amyl, hexyl, decyl and the like.

The term “alkenyl”, alone or in combination, refers to a straight-chainor branched-chain alkenyl radical containing from 2 to 10, preferablyfrom 2 to 6 and more preferably from 2 to 4, carbon atoms. Examples ofsuch radicals include, but are not limited to, ethenyl, E- andZ-propenyl, isopropenyl, E- and Z-butenyl, E- and Z-isobutenyl, E- andZ-pentenyl, decenyl and the like.

The term “alkynyl”, alone or in combination, refers to a straight-chainor branched-chain alkynyl radical containing from 2 to 10, preferablyfrom 2 to 6 and more preferably from 2 to 4, carbon atoms. Examples ofsuch radicals include, but are not limited to, ethynyl (acetylenyl),propynyl, propargyl, butynyl, hexynyl, decynyl and the like.

The term “cycloalkyl”, alone or in combination, refers to a cyclic alkylradical containing from 3 to 8, preferably from 3 to 6, carbon atoms.Examples of such cycloalkyl radicals include, but are not limited to,cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl and the like.

The term “cycloalkenyl”, alone or in combination, refers to a cycliccarbocycle containing from 4 to 8, preferably 5 or 6, carbon atoms andone or more double bonds. Examples of such cycloalkenyl radicalsinclude, but are not limited to, cyclopentenyl, cyclohexenyl,cyclopentadienyl and the like.

The term “aryl” refers to a carbocyclic aromatic group selected from thegroup consisting of phenyl, naphthyl, indenyl, indanyl, azulenyl,fluorenyl, and anthracenyl; or a heterocyclic aromatic group selectedfrom the group consisting of furyl, thienyl, pyridyl, pyrrolyl,oxazolyly, thiazolyl, imidazolyl, pyrazolyl, 2-pyrazolinyl,pyrazolidinyl, isoxazolyl, isothiazolyl, 1,2,3-oxadiazolyl,1,2,3-triazolyl, 1,3,4-thiadiazolyl, pyridazinyl, pyrimidinyl,pyrazinyl, 1,3,5-triazinyl, 1,3,5-trithianyl, indolizinyl, indolyl,isoindolyl, 3H-indolyl, indolinyl, benzo[b]furanyl,2,3-dihydrobenzofuranyl, benzo[b]thiophenyl, 1H-indazolyl,benzimidazolyl, benzthiazolyl, purinyl, 4H-quinolizinyl, quinolinyl,isoquinolinyl, cinnolinyl, phthalazinyl, quinazolinyl, quinoxalinyl,1,8-naphthyridinyl, pteridinyl, carbazolyl, acridinyl, phenazinyl,phenothiazinyl, and phenoxazinyl.

“Aryl” groups, as defined in this application may independently containone to four substituents which are independently selected from the groupconsisting of hydrogen, halogen, hydroxyl, amino, nitro,trifluoromethyl, trifluoromethoxy, alkyl, alkenyl, alkynyl, cyano,carboxy, carboalkoxy, Ar′-substituted alkyl, Ar′-substituted alkenyl oralkynyl, 1,2-dioxymethylene, 1,2-dioxyethylene, alkoxy, alkenoxy oralkynoxy, Ar′-substituted alkoxy, Ar′-substituted alkenoxy or alkynoxy,alkylamino, alkenylamino or alkynylamino, Ar′-substituted alkylamino,Ar′-substituted alkenylamino or alkynylamino, Ar′-substitutedcarbonyloxy, alkylcarbonyloxy, aliphatic or aromatic acyl,Ar′-substituted acyl, Ar′-substituted alkylcarbonyloxy, Ar′-substitutedcarbonylamino, Ar′-substituted amino, Ar′-substituted oxy,Ar′-substituted carbonyl, alkylcarbonylamino, Ar′-substitutedalkylcarbonylamino, alkoxy-carbonylamino, Ar′-substitutedalkoxycarbonyl-amino, Ar′-oxycarbonylamino, alkylsulfonylamino, mono- orbis-(Ar′-sulfonyl)amino, Ar′-substituted alkyl-sulfonylamino,morpholinocarbonylamino, thiomorpholinocarbonylamino, N-alkyl guanidino,N-Ar′ guanidino, N-N-(Ar′, alkyl) guanidino, N,N-(Ar′,Ar′)guanidino,N,N-dialkyl guanidino, N,N,N-trialkyl guanidino, N-alkyl urea,N,N-dialkyl urea, N-Ar′ urea, N,N-(Ar′,alkyl) urea and N,N-(Ar′)₂ urea;acylcarbonylamino; Ar′-substituted aryl; aromatic acyl-substitutedaromatic or aliphatic acyl; Ar′-substituted heterocyclyl;Ar′-substituted cycloalkyl or cycloalkenyl; heterocyclylalkoxy;N,N-(Ar′, hydroxyl)urea;Ar′-substituted cycloalkyl and cycloalkenyl;Ar′-substituted biaryl; Ar′-substituted aminocarbonylamino;Ar′-mercapto-substituted alkyl; Ar′-amino-substituted aryl;Ar′-oxysubstituted alkyl; Ar′-substituted aminocycloalkyl andcycloalkenyl; aralkylaminosulfonyl; aralkoxyalkyl; N-Ar′-substitutedthiourea; N-aralkoxyurea; N-hydroxylurea; N-alkenylurea; N,N-(alkyl,hydroxyl)urea; heterocyclyl; thioaryloxy-substituted aryl;N,N-(aryl,alkyl)hydrazino; Ar′-substituted sulfonylheterocyclyl;aralkyl-substituted heterocyclyl; cycloalkyl and cycloakenyl-substitutedheterocyclyl; cycloalkyl-fused aryl; aryloxy-substituted alkyl;heterocyclylamino; Ar′-substituted arylaminosulfonyl;thioaryl-substituted thioxy; and Ar′-substituted alkenoyl; aliphatic oraromatic acylaminocarbonyl; aliphatic or aromatic acyl-substitutedalkenyl; Ar′-substituted aminocarbonyloxy; Ar′,Ar′-disubstituted aryl;aliphatic or aromatic acyl-substituted acyl;benzofused-heterocyclylcarbonylamino; Ar′-substituted hydrazino;Ar′-substituted aminosulfonyl; Ar′-substituted alkylimino;Ar′-substituted heterocyclyl; Ar′,Ar′-disubstituted acylamino;Ar′-substituted cycloalkenonylamino; heterocyclylalkoxy;N,N-Ar′,hydroxylurea; N,N′-Ar′,hydroxylurea; heterocyclylcarbonylamino;Ar′-substituted aminocarbonylheterocyclyl; Ar′-substitutedaminocarbonyl; Ar′-substituted carbonylamino; Ar′-substitutedaminosulfonylamino; Ar′-substituted mercaptoalkyl; Ar′-amino substitutedbiaryl; aralkylaminoalkoxy; alkyl- and aryloxy-substituted alkoxy;heterocyclylcarbonyl; Ar′-substituted sulfonylalkyl; Ar′-aminocarbocyclyl; aralkylsulfonyl; aryl-substituted alkenyl;heterocyclylalkylamino; heterocyclylalkylaminocarbonyl; Ar′-substitutedsulfonylaminoalkyl; Ar′-substituted cycloalkyl; thioaryloxyalkyl;thioaryloxymercapto; cycloalkylcarbonylalkyl; cycloalkyl-substitutedamino; Ar′-substituted arylamino; aryloxycarbonylalkyl;phosphorodiamidyl acid or ester; aryloxydimethylsiloxy;1,3-indandionylcarbonylalkyl; 1,3-indandionylcarbonyl; oxamidyl;heterocyclylalkylidenyl; formamidinyl; benzalizinyl; benzalhydrazino;arylsulfonylurea; benzilylamino;4-(N-2-carboxyalkyl-1-(1,3-benzodioxol-5-yl)-amino-N-leucinylalkylamidylarylurea);Ar′-carbamoyloxy and alkyl- and aryloxy-substituted urea; wherein “Ar′”is a carbocyclic or heterocyclic aryl group as defined above having oneto three substituents selected from the group consisting of hydrogen,halogen, hydroxyl, amino, nitro, trifluoromethyl, trifluoromethoxy,alkyl, alkenyl, alkynyl, 1,2-dioxymethylene, 1,2-dioxyethylene, alkoxy,alkenoxy, alkynoxy, alkylamino, alkenylamino or alkynylamino,alkylcarbonyloxy, aliphatic or aromatic acyl, alkylcarbonylamino,alkoxycarbonylamino, alkylsulfonylamino, N-alkyl or N,N-dialkyl urea.

The term “alkoxy”, alone or in combination, refers to an alkyl etherradical, wherein the term “alkyl” is as defined above. Examples ofsuitable alkyl ether radicals include, but are not limited to, methoxy,ethoxy, n-propoxy, iso-propoxy, n-butoxy, iso-butoxy, sec-butoxy,tert-butoxy and the like.

The term “alkenoxy”, alone or in combination, refers to a radical offormula alkenyl-O—, wherein the term “alkenyl” is as defined aboveprovided that the radical is not an enol ether. Examples of suitablealkenoxy radicals include, but are not limited to, allyloxy, E- andZ-3-methyl-2-propenoxy and the like.

The term “alkynyloxy”, alone or in combination, refers to a radical offormula alkynyl-O—, wherein the term “alkynyl” is as defined aboveprovided that the radical is not an ynol ether. Examples of suitablealkynoxy radicals include, but are not limited to, propargyloxy,2-butynyloxy and the like.

The term “thioalkoxyl” refers to a thioether radical of formulaalkyl-S—, wherein alkyl is as defined above.

The term “alkylamino”, alone or in combination, refers to a mono- ordi-alkyl-substituted amino radical (i.e., a radical of formula alkyl-NH—or (alkyl)₂-N—), wherein the term “alkyl” is as defined above. Examplesof suitable alkylamino radicals include, but are not limited to,methylamino, ethylamino, propylamino, isopropylamino, t-butylamino,N,N-diethylamino and the like.

The term “alkenylamino”, alone or in combination, refers to a radical offormula alkenyl-NH— or (alkenyl)₂N—, wherein the term “alkenyl” is asdefined above, provided that the radical is not an enamine. An exampleof such alkenylamino radicals is the allylamino radical.

The term “alkynylamino”, alone or in combination, refers to a radical offormula alkynyl-NH— or (alkynyl)₂N—, wherein the term “alkynyl” is asdefined above, provided that the radical is not an ynamine. An exampleof such alkynylamino radicals is the propargyl amino radical.

The term “aryloxy”, alone or in combination, refers to a radical offormula aryl-O—, wherein aryl is as defined above. Examples of aryloxyradicals include, but are not limited to, phenoxy, naphthoxy, pyridyloxyand the like.

The term “arylamino”, alone or in combination, refers to a radical offormula aryl-NH—, wherein aryl is as defined above. Examples ofarylamino radicals include, but are not limited to, phenylamino(anilido), naphthylamino, 2-, 3- and 4-pyridylamino and the like.

The term “biaryl”, alone or in combination, refers to a radical offormula aryl-aryl-, wherein the term “aryl” is as defined above.

The term “thioaryl”, alone or in combination, refers to a radical offormula aryl-S—, wherein the term “aryl” is as defined above. An exampleof a thioaryl radical is the thiophenyl radical.

The term “aryl-fused cycloalkyl”, alone or in combination, refers to acycloalkyl radical which shares two adjacent atoms with an aryl radical,wherein the terms “cycloalkyl” and “aryl” are as defined above. Anexample of an aryl-fused cycloalkyl radical is the benzofused cyclobutylradical.

The term “aliphatic acyl”, alone or in combination, refers to radicalsof formula alkyl-CO—, alkenyl-CO— and alkynyl-CO— derived from analkane-, alkene- or alkyncarboxylic acid, wherein the terms “alkyl”,“alkenyl” and “alkynyl” are as defined above. Examples of such aliphaticacyl radicals include, but are not limited to, acetyl, propionyl,butyryl, valeryl, 4-methylvaleryl, acryloyl, crotyl, propiolyl,methylpropiolyl and the like.

The term “aromatic acyl”, alone or in combination, refers to a radicalof formula aryl-CO—, wherein the term “aryl” is as defined above.Examples of suitable aromatic acyl radicals include, but are not limitedto, benzoyl, 4-halobenzoyl, 4-carboxybenzoyl, naphthoyl, pyridylcarbonyland the like.

The terms “morpholinocarbonyl” and “thiomorpholinocarbonyl”, alone or incombination with other terms, refer to an N-carbonylated morpholino andan N-carbonylated thiomorpholino radical, respectively.

The term “alkylcarbonylamino”, alone or in combination, refers to aradical of formula alkyl-CONH, wherein the term “alkyl” is as definedabove.

The term “alkoxycarbonylamino”, alone or in combination, refers to aradical of formula alkyl-OCONH—, wherein the term “alkyl” is as definedabove.

The term “alkylsulfonylamino”, alone or in combination, refers to aradical of formula alkyl-SO₂NH—, wherein the term “alkyl” is as definedabove.

The term “arylsulfonylamino”, alone or in combination, refers to aradical of formula aryl-SO₂NH—, wherein the term “aryl” is as definedabove.

The term “N-alkylurea”, alone or in combination, refers to a radical offormula alkyl-NH—CO—NH—, wherein the term “alkyl” is as defined above.

The term “N-arylurea”, alone or in combination, refers to a radical offormula aryl-NH—CO— NH—, wherein the term “aryl” is as defined above.

The term “halogen” means fluorine, chlorine, bromine and iodine.

The term “heterocycle” (and corresponding “heterocyclyl” radical form)unless otherwise defined herein, refers to a stable 3-7 memberedmonocyclic heterocyclic ring or 8-11 membered bicyclic heterocyclic ringwhich is unsaturated, and which may be optionally benzofused. Eachheterocycle consists of one or more carbon atoms and from one to fourheteroatoms selected from the group consisting of nitrogen, oxygen andsulfur. As used herein, the terms “nitrogen and sulfur heteroatoms”include any oxidized form of nitrogen and sulfur, and the quaternizedform of any basic nitrogen. In addition, any ring nitrogen may beoptionally substituted with a substituent R⁴, as defined herein forcompounds of formula I. A heterocycle may be attached at any endocycliccarbon or heteroatom which results in the creation of a stablestructure. Preferred heterocycles include 5-7 membered monocyclicheterocycles and 8-10 membered bicyclic heterocycles. Heterocycles maybe optionally oxo-substituted at 1-3 ring positions and may optionallybe independently substituted with 1-4 substituents selected from thegroup of “aryl” substituents described above.

The term “leaving group” generally refers to groups readily displaceableby a nucleophile, such as an amine, and alcohol or a thiol nucleophile.Such leaving groups are well known and include carboxylates,N-hydroxysuccinimide, N-hydroxybenzotriazole, halogen (halides),triflates, tosylates, mesylates, alkoxy, thioalkoxy and the like.

The terms “activated derivative of a suitably protected a-amino acid”and “activated substituted-phenylacetic acid derivative” refer to thecorresponding acyl halides (e.g. acid fluoride, acid chloride and acidbromide), corresponding activated esters (e.g. nitrophenyl ester, theester of 1-hydroxybenzotriazole, HOBT, or the ester ofhydroxysuccinimide, HOSu), and other conventional derivatives within theskill of the art.

In view of the above definitions, other chemical terms used throughoutthis application can be easily understood by those of skill in the art.Terms may be used alone or in any combination thereof. The preferred andmore preferred chain lengths of the radicals apply to all suchcombinations.

This invention provides compounds which are capable of inhibitingVLA-4-mediated cell adhesion by inhibiting the binding of ligands tothat receptor. These compounds are represented by formula (I):

and pharmaceutically acceptable derivatives thereof;

wherein:

X is selected from the group consisting of —CO₂H, —PO⁻ ₃H, —SO₂R₅,—SO₃H, —OPO⁻ ₃H, —CO₂R₄ and —C(O)N(R₄)₂;

wherein R₅ is selected from the group consisting of alkyl, alkenyl,alkynyl, cycloalkyl, cycloalkenyl, aryl, aryl-substituted alkyl, andaryl-substituted alkenyl or alkynyl;

Y is selected from the group consisting of —CO—, —SO₂— and —PO₂—;

R₁ is selected from the group consisting of alkyl, alkenyl, alkynyl,cycloalkyl, aryl-fused cycloalkyl, cycloalkenyl, aryl, aryl-substitutedalkyl (“aralkyl”), aryl-substituted alkenyl or alkynyl,cycloalkyl-substituted alkyl, cycloalkenyl-substituted cycloalkyl,biaryl, alkoxy, alkenoxy, alkynoxy, aryl-substituted alkoxy(“aralkoxy”), aryl-substituted alkenoxy or alkynoxy, alkylamino,alkenylamino or alkynylamino, aryl-substituted alkylamino,aryl-substituted alkenylamino or alkynylamino, aryloxy, arylamino,N-alkylurea-substituted alkyl, N-arylurea-substituted alkyl,alkylcarbonylamino-substituted alkyl, aminocarbonyl-substituted alkyl,heterocyclyl, heterocyclyl-substituted alkyl, heterocyclyl-substitutedamino, carboxyalkyl substituted aralkyl, oxocarbocyclyl-fused aryl andheterocyclylalkyl;

R₂ is selected from the group consisting of hydrogen, aryl, alkyl,alkenyl or alkynyl, cycloalkyl, cycloalkenyl, aryl-substituted alkyl andwherein R₂ and R₃ may be taken together with the atoms to which they areattached, to form a heterocycle;

R₃ is selected from the group consisting of alkyl, alkenyl, alkynyl,cycloalkyl, cycloalkenyl, aralkyl, aryl-substituted alkenyl or alkynyl,hydroxy-substituted alkyl, alkoxy-substituted alkyl,aralkoxy-substituted alkyl, amino-substituted alkyl, (aryl-substitutedalkyloxycarbonylamino)-substituted alkyl, thiol-substituted alkyl,alkylsulfonyl-substituted alkyl, (hydroxy-substitutedalkylthio)-substituted alkyl, thioalkoxy-substituted alkyl,acylamino-substituted alkyl, alkylsulfonylamino-substituted alkyl,arylsulfonylamino-substituted alkyl, morpholino-alkyl,thiomorpholino-alkyl, morpholino carbonyl-substituted alkyl,thiomorpholinocarbonyl-substituted alkyl, [N-(alkyl, alkenyl oralkynyl)- or N,N-[dialkyl, dialkenyl, dialkynyl or(alkyl,alkenyl)-amino]carbonyl-substituted alkyl, carboxyl-substitutedalkyl, dialkylamino-substituted acylaminoalkyl and amino acid sidechains selected from arginine, asparagine, glutamine, S-methyl cysteine,methionine and corresponding sulfoxide and sulfone derivatives thereof,glycine, leucine, isoleucine, allo-isoleucine, tert-leucine, norleucine,phenylalanine, tyrosine, tryptophan, proline, alanine, ornithine,histidine, glutamine, valine, threonine, serine, aspartic acid,beta-cyanoalanine, and allothreonine,wherein R₂ and R₃ may be takentogether with the atoms to which they are attached, to form aheterocycle;

R₄ is selected from the group consisting of aryl, alkyl, cycloalkyl,alkenyl, cycloalkenyl, alkynyl and aryl-substituted alkyl, hydrogen,heterocyclyl, heterocyclylcarbonyl, aminocarbonyl, amido, mono- ordialkylaminocarbonyl, mono- or diarylaminocarbonyl,alkylarylaminocarbonyl, diarylaminocarbonyl, mono- ordiacylaminocarbonyl, aromatic or aliphatic acyl, alkyl optionallysubstituted by substituents selected from the group consisting of amino,hydroxy, mercapto, mono- or dialkylamino, mono- or diarylamino,alkylarylamino, diarylamino, mono- or diacylamino, alkoxy, alkenoxy,aryloxy, thioalkoxy, thioalkenoxy, thioalkynoxy, thioaryloxy andheterocyclyl; and

n is 0, 1 or 2.

A “pharmaceutically acceptable derivative” denotes any pharmaceuticallyacceptable salt, ester, or salt of such ester, of a compound of thisinvention. The invention also includes any other compound which, uponadministration to a patient, is capable of providing (directly orindirectly) a compound of this invention (e.g. a prodrug). The inventionalso includes metabolites or residues of a compound of this inventioncharacterized by the ability to inhibit, prevent or suppress celladhesion and cell adhesion-mediated pathologies.

In another preferred embodiment of this invention, R₁ is selected fromthe group consisting of benzyloxy, cyanomethyl, cyclohexylmethyl,methyl, n-hexyl, N-phenylamino, phenyl, phenylcarbonyl, phenylmethyl,t-butoxy, t-butylamino, 1-indanyl, 1-naphthylmethyl,1-phenylcyclopropyl, 2-(4-hydroxyl-phenyl)ethyl,2-(benzyloxycarbonylamino)-phenylmethyl,2-(bis(phenyl-sulfonyl)amino)-phenylmethyl,2-(N′-phenylurea)phenyl-methyl, 2-aminophenylmethyl,2-benzamidophenylmethyl, 2-bromo-4-hydroxy-5-methoxyphenylmethyl,2-hydroxyphenyl-methyl, 2-naphthylmethyl, 2-phenylethyl,2-pyridylmethyl, 2-quinolinyl, 2-[4-(N′-phenylurea)phenyl]-ethyl,3-(benzyloxycarbonylamino)-phenylmethyl, 3-(N′-phenylurea)-phenylmethyl,3-(N′-phenylurea)propyl, 3-(phenylsulfon-amido)-phenylmethyl,3-acetamidophenylmethyl, 3-amino-phenylmethyl, 3-benzamidophenylmethyl,3-hydroxy-4-(N′-phenylurea)-phenylmethyl, 3-hydroxyphenylmethyl,3-indolyl, 3-methyoxy-4-(N′-phenylurea)-phenylmethyl,3-methoxy-4-(N′-(2-methylphenyl)urea)-phenylmethyl,3-methyl-4-(N′-phenylurea)-phenylmethyl, 3-nitrophenylmethyl,3-phenylpropyl, 3-pyridylmethyl, 4-(2-aminobenzamido)-phenylmethyl,4-(benzamido)phenylmethyl, 4-(benzyloxycarbonylamino)-phenylmethyl,4-(morpholinocarbonylamino)-phenylmethyl,4-(N′-(2-chlorophenyl)urea)-phenylmethyl,4-(N′-(2-chlorophenyl)urea)-3-methoxyphenylmethyl,4-(N′-(2-ethylphenyl)urea)-phenylmethyl,4-(N′-(2-isopropylphenyl)urea)-phenylmethyl,4-(N′-(2-methoxyphenyl)urea)phenylmethyl,4-(N′-(2-methyl-3-pyridyl)urea)-phenylmethyl,4-(N′-(2-nitrophenyl)urea)-phenylmethyl,4-(N′-(2-pyridyl)urea)-phenylmethyl,4-(N′-(2-t-butylphenyl)-urea)-phenylmethyl,4-(N′-(2-thiazolyl)urea)-phenylmethyl,4-(N′-(3-chlorophenyl)urea)-phenylmethyl,4-(N′-(3-methoxyphenyl)urea)-phenylmethyl,4-(N′-(3-pyridyl)-urea)-phenylmethyl,4-(N′-(4-pyridyl)urea)-phenylmethyl,4-(N′-(3-methylphenyl)urea)-phenylmethyl,4-(N′-(2-methylphenyl)-urea)-phenylmethyl,4-(N′-benzylurea)phenylmethyl, 4-(N′-cyclohexylurea)-phenylmethyl,4-(N′-ethylurea)-phenylmethyl, 4-(N′-isopropylurea)-phenylmethyl,4-(N′-methylurea)phenylmethyl, 4-(N′-p-toluylurea)-phenyl-methyl,4-(N′-phenylurea)phenyl, 4-(N′-phenylurea)phenyl-amino,4-(N′-phenylurea)phenylmethyl, 4-(N′-t-butylurea)-phenylmethyl,4-(phenylaminocarbonylamino-methyl)-phenyl,4-(phenylsulfonamido)-phenylmethyl,4-(t-butoxycarbonylamino)-phenylmethyl, 4-acetamidophenylmethyl,4-aminophenylamino, 4-amino-phenylmethyl, 4-benzamidophenylmethyl,4-chlorophenylmethyl, 4-hydroxy-3-nitrophenylmethyl,4-hydroxyphenylmethyl, 4-methoxyphenylmethyl, 4-nitrophenylamino,4-nitrophenylmethyl, 4-phenacetamidophenylmethyl, 4-phenylphenylmethyl,4-pyridylmethyl, 4-trifluoromethylphenylmethyl,4-[2-(N′-methylurea)benzamido]-phenylmethyl,4-(N-′-(2-methylphenyl)urea)phenyl-methyl,4-(N′-phenyl-N″-methylguanidino)phenyl-methyl, 5-(N′-phenylurea)pentyl,5-(N′-t-butylurea)-pentyl, 2,2-dimethylpropyl, 2,2-diphenylmethyl,2,3-benzocyclobutyl, 3,4-dihydroxyphenylmethyl,3,5-dimethoxy-4-hydroxy-phenylmethyl,4-(1-indolecarboxylamino)-phenylmethyl,6-methoxy-5-(N′-(2-methylphenyl)urea)-2-pyridylmethyl,4-(1,3-benzoxazol-2-ylamino)-phenylmethyl and4-(1,3-imdazol-2-ylamino)-phenylmethyl, 3-carboxy-1-phenylpropyl;3-hydroxy-4-(2-methylphenyl)ureaphenylmethyl;3-hydroxy-4-(2-chlorophenyl)ureaphenylmethyl; 6-(phenylurea)heptyl,4-phenylurea)butyl; 2-thienylmethyl;4-(2,6-dimethylphenylurea)phenylmethyl;4-(2-hydroxyphenylurea)phenylmethyl;3-butoxy-4-(2-methylphenyl)ureaphenylmethyl;3-butoxy-4-(phenylurea)phenylmethyl; 4-(N-2-pyrazinylurea)phenylmethyl;2-phenylethynyl; 5-phenylurea-2-pyridylmethyl;5-(2-methylphenylurea)-2-pyridylmethyl;4-(3-methyl-2-pyridylurea)phenylmethyl;3-nitro-4-(phenylurea)phenylmethyl;3-acylamino-4-(phenylurea)phenylmethyl; 4-(N,N-phenyl,methylurea)phenylmethyl; 4-(3-hydroxyphenylurea)phenylmethyl;4-(2-acetylaminophenylurea)phenylmethyl;4-(2-propionylaminophenylurea)phenylmethyl;4-(3-benzyloxy-2-pyridylurea)phenylmethyl;4-(3-methyl-2-pyridylurea)phenylmethyl;4-(indolylcarbonylamino)phenylmethyl; 2-(4-(phenylurea)phenyl)oxiranyl;4-(N,N′-phenyl, methylurea)phenylmethyl;4-(2-dimethylaminophenylurea)phenylmethyl;4-(2-benzimidazolylamino)phenylmethyl;4-(2-benzoxazolylamino)phenylmethyl;4-(2-benzthiazolylamino)phenylmethyl;4-(tetrahydroquinolinylcarbonylamino)phenylmethyl;1,3-dimethyl-3-(phenylurea)butyl; hydroxyethylthiomethyl;4-(phenylurea)phenylethenyl; 3-amino-4-(phenylurea)phenylmethyl;4-(4-hydroxyphenylurea)phenylmethyl; 4-(2-aminophenylurea)phenylmethyl;4-((2-methylurea)phenylurea)phenyl;4-(2-hydroxyphenylurea)-3-methoxyphenylmethyl;4-(2-methylsulfonylmethylphenylurea)phenylmethyl;4-(2-methylphenylurea)tetrahydro-2-pyrimidonylmethyl;3-methoxy-4-(phenylurea)-2-pyridylmethyl;4-(2-trifluoromethylphenylurea)phenylmethyl;4-(3-methyl-2-pyridylurea)phenylmethyl;4-(2,4(1H,3H)-quinazolinedionyl)phenylmethyl; 4-thioureaphenylmethyl;4-(phenylthiourea)phenylmethyl;4-(pyrrolidinylcarbonylamino)phenylmethyl;4-(2-benzoxazolinonylcarbonylamino)phenylmethyl;4-(benzyloxyurea)phenylmethyl;4-(thiazolidinylcarbonylamino)phenylmethyl; 4-benzoylureaphenylmethyl;hydroxylureaphenylmethyl; N′,N′-methyl,hydroxylureaphenylmethyl;4-(N′-allylurea)phenylmethyl;4-(3-pyrrolidinylcarbonylamino)phenylmethyl;4-(1-pyrrolylcarbonylamino)phenylmethyl;4-(2-pyrrolylcarbonylamino)phenylmethyl; 4-(propylurea)phenylmethyl;4-(methoxyurea)phenylmethyl; 4-(dimethylurea)phenylmethyl;4-(2-quinazolinylamino)phenylmethyl; 4-(2-furanoylamino)phenylmethyl;4-(2-hydroxy-6-methylphenylurea)phenylmethyl;4-(2-pyridylcarbonylamino)phenylmethyl;4-(3-hydroxy-2-methylphenylurea)phenylmethyl;4-(2-fluorophenylurea)phenylmethyl; 4-(3-fluorophenylurea)phenylmethyl;4-(4-fluorophenylurea)phenylmethyl;4-(2-quinolinylcarbonylamino)phenylmethyl;4-(isoquinolinylcarbonylamino)phenylmethyl;4-(2,3-dimethylphenylurea)phenylmethyl;4-(2,5-dimethylphenylurea)phenylmethyl;4-(2-methyl-4-fluorophenylurea)phenylmethyl;4-(2-methyl-3-fluorophenylurea)phenylmethyl; 3-carboxy-3-phenylpropyl;4-(5-hydroxy-2-methyphenylurea)phenylmethyl;4-(4-hydroxy-2-methylphenylurea)phenylmethyl;4-(2,4-difluorophenylurea)phenylmethyl; 3-dibenzofuranylcarbonyl;4-(phenoxycarbonylamino)phenylmethyl; 3-phenylureapropyl;4-(phenylaminocarbonyloxy)phenylmethyl; 4-cinnamoylphenylmethyl;dibenzofuranylmethyl; 4-(2-methylphenylaminocarbonyloxy)phenylmethyl;methylphenylurea)phenylamino; 4-(3-indolylcarbonylamino)phenylmethyl;4-(phenylaminocarbonyl)phenylmethyl; 4-phenylalkynylphenylmethyl;4-(3-pyrrolylcarbonylamino)phenylmethyl; 5-nitrobenzofuran-2-yl;5-(2-methylphenylurea)benzofuran-2-yl; 3-carboxy-3-phenylpropyl;2-(3-pyridyl)-thiazol-4-yl; 2-(4-pyridyl)-thiazol-4-yl; 2-oxo- and4-oxo-4,5,6,7-tetrahydrobenzo[b]furan-3-yl;3-methoxy-4-(phenylcarbamoyloxy)phenylmethyl; 5-amino-benzofuran-2-yl;benzilylaminophenylmethyl and4-[N-2-carboxyethyl-1-(1,3-benzodioxolyl-5-yl)amino-N-leucinylacetamidylphenylurea]phenylmethyl.

Most preferably, R₁ is selected from the group consisting of4-hydroxyphenylmethyl, 3-methoxy-4-(N′-phenylurea)-phenylmethyl,4-(N′-phenylurea)-phenylmethyl, 4-(N′-(2-methylphenyl)urea)phenylmethyl,4-(N′-2-pyridyl)-urea)-phenylmethyl,3-methoxy-4-(N′-(2-methylphenyl)urea)phenylmethyl,6-methoxy-5-(N′-(2-methylphenyl)urea)-2-pyridylmethyl,4-(N′-3-methyl-2-pyridylurea)phenylmethyl,3-methoxy-4-(N′-3-methyl-2-pyridylurea)phenylmethyl, and3-methoxy-4-(N′-2-pyridylurea)phenylmethyl.

In an alternate preferred embodiment, R₁ is an aryl-substituted C₁-C₄alkyl group. More preferably, R₁ is a (N-Ar′-urea)-para-substitutedarylalkyl group, and most preferably, a (N-Ar′-urea)-para-substitutedphenylmethyl group.

According to another preferred embodiment, R₂ is selected from the groupconsisting of hydrogen, methyl or phenacetyl. Most preferably, R₂ ishydrogen.

According to another preferred embodiment, R₃ is selected from the groupconsisting of 2-(methylsulfonyl)-ethyl, 3-(hyrdoxypropylthio)-methyl,4-(methylsulfonylamino)-butyl, 4-acetylaminobutyl, aminomethyl, benzyl,butyl, hydroxymethyl, isobutyl, methyl, methylthiomethyl, phenylmethyl,propyl, 4-(benzyloxycarbonylamino)-butyl, N,N-(methylpropargyl)amino,2-(methylthio)-ethyl, 2-(morpholino-N-carbonyl)-ethyl,2-(N-morpholino)-ethyl, 2-(N,N-dimethylamino)-ethyl, 4-amino-butyl,4-benzyloxyphenylmethyl, 2-benzylthiomethyl,t-butoxycarbonylaminomethyl, sec-butyl, t-butyl,N,N-dimethylaminocarbonylmethyl, 1,1-ethano,* 4-hydroxyphenylmethyl,1-hydroxyethyl, 1-methoxyethyl, 4-methoxyphenylmethyl, benzyloxy-methyl,

* The amino acid side chain derived from 1-aminocyclopropylcarboxylicacid. benzylthio-methyl, carbonylmethyl, 2-methylsulfinylethyl,morpholino-N-carbonylmethyl, thiomorpholino-N-carbonylmethyl,2-phenylethyl, asparagine side-chain, proline side-chain,2-thiazolyl-methyl, 4-(phenylurea)butyl; 4-(methylurea)butyl;morpholinocarbonylmethylthiomethyl; morpholinoethylthiomethyl;3-pyridylmethyl; 4-methylsulfonylaminobutyl; hydroxymethylthiomethyl;2-methylsulfonylethyl, 4-propionylaminobutyl;4-ethoxycarbonylaminobutyl; methoxycarbonylaminobutyl;carbomethoxymethylthiomethyl; 4-t-butylureabutyl;carboxymethylthiomethyl; dimethylamidomethylthiomethyl;acetylaminopropyl; 3-methylureapropyl; 4-biotinoylaminobutyl;2-thienylmethyl; 3-pyridylmethyl; 4-trifluoroacetylaminobutyl;dimethylaminomethylthiomethyl; dimethylaminoethylthiomethyl;4-(dimethylaminoacetylamino)butyl or in combination with R₂ forms aproline, azetidine or pipecolinic ring.

Most preferably, R₃ is selected from the group consisting of isobutyl,2-(methylthio)-ethyl, 3-(hydroxypropylthio)-methyl,2-(methylsulfonyl)-ethyl, 4-acetylamino-butyl,4-(methylsulfonylamino)-butyl, and 4-(ethoxycarbonylamino)butyl.

According to yet another embodiment, R₄ is selected from the groupconsisting of 4-carbomethoxyphenyl, 4-carboxyphenyl, 4-fluorophenyl,4-methoxyphenyl, benzyl, methyl, phenyl, phenylmethyl, phenylethyl,4-chlorophenyl, 3,4-difluorophenyl, 3,4-dimethoxyphenyl,2-methoxyphenyl, 3-methoxyphenyl, 4-methoxyphenyl, 2-nitrophenyl,3-pyridyl, 4-phenoxyphenyl; 4-ethoxyphenyl; 4-nitrophenyl;4-acetylaminophenyl; 4-methylureaphenyl; 2-fluorophenyl; naphthyl;3-fluorophenyl; 3-nitrophenyl; hydrogen; 2-nitrophenyl; 4-cyanophenyl;3-methoxyphenyl; 4-methylsulfonylamino; 3-cyanophenyl; 4-propionylamino;4-aminophenyl; 3-aminophenyl; 4-trifluoromethoxyphenyl; 4-methylphenyl;4-amino-3-nitrophenyl; 4-hydroxy-3-methoxyphenyl; 4-hexyloxyphenyl;4-methylthiophenyl; 3-furanyl; 4-dimethylaminophenyl;3-hydroxy-4-nitrophenyl; n-pentyl; carboxymethyl; 2-carboxyethyl;ethynyl; 2-thienyl; 2-propenyl; 2-propynyl; methyl; and propyl. Morepreferably, R₄ is selected from the group consisting 4-methoxyphenyl,3,4-dimethoxyphenyl, 4-fluorophenyl, 4-carboxyphenyl,4-carbomethoxyphenyl, phenylethyl, phenylmethyl, allyl, ethynyl, and3,4-methylenedioxyphenyl.

In another preferred embodiment Y is CO, CH₂ or SO₂. Most preferably, Yis CO.

According to another preferred embodiment, X in formula (I) is COOH.

According to yet another preferred embodiment, n is 1.

Examples of some preferred compounds of this invention wherein X is acarboxyl group and n is 1 are provided in Table 1.

TABLE 1

Bio # R¹ R² R³ R⁴ Y 1002 cyanomethyl H isobutyl phenyl CO 1003cyclohexylmethyl H isobutyl phenyl CO 1004 2-pyridylmethyl H isobutylphenyl CO 1005 3-pyridylmethyl H isobutyl phenyl CO 10064-hydroxyphenylmethyl H isobutyl 1,3-benzo- CO dioxol-5-yl 10074-pyridylmethyl H isobutyl phenyl CO 1008 phenyl H isobutyl phenyl CO1009 4-phenylphenylmethyl H isobutyl phenyl CO 1010 4-chlorophenylmethylH isobutyl phenyl CO 1011 4-trifluoromethylphenylmethyl H isobutylphenyl CO 1013 phenylmethyl H isobutyl phenyl SO₂ 1014 3-indolyl Hisobutyl phenyl CO 1015 4-benzamidophenylmethyl H isobutyl phenyl CO1016 4-aminophenylmethyl H isobutyl phenyl CO 1017 1-phenylcyclopropyl Hisobutyl phenyl CO 1018 3-acetamidophenylmethyl H isobutyl phenyl CO1020 3-benzamidophenylmethyl H isobutyl phenyl CO 1021 1-naphthylmethylH isobutyl phenyl CO 1022 2-naphthylmethyl H isobutyl phenyl CO 10234-phenacetamidophenylmethyl H isobutyl phenyl CO 10242-aminophenylmethyl H isobutyl phenyl CO 10252-(bis(phenylsulfonyl)amino)- H isobutyl phenyl CO phenylmethyl 10262-benzamidophenylmethyl H isobutyl phenyl CO 10272-(benzyloxycarbonylamino)- H isobutyl phenyl CO phenylmethyl 10284-(2-aminobenzamido)- H isobutyl phenyl CO phenylmethyl 10294-[2-(N′-methylurea)-benzamido]- H isobutyl phenyl CO phenylmethyl 10303-aminophenylmethyl H isobutyl phenyl CO 10313-(benzyloxycarbonylamino)- H isobutyl phenyl CO phenylmethyl 10323-(phenylsulfonamido)- H isobutyl phenyl CO phenylmethyl 1036phenylmethyl H isobutyl 1,3-benzo- CO dioxol-5-yl 10374-(N′-phenylurea)- H 2-thiazolyl- phenyl CO phenylmethyl methyl 1038phenylmethyl H propyl phenyl CO 1039 phenylmethyl H butyl phenyl CO 1040phenylmethyl H sec-butyl phenyl CO 1041 t-butoxy H hydroxymethyl phenylCO 1042 t-buyoxy H phenylmethyl phenyl CO 1043 t-butoxy H 1,1-ethanophenyl CO 1044 t-butoxy methyl isobutyl phenyl CO 1045 phenylmethyl Hhydroxymethyl phenyl CO 1046 phenylmethyl H phenylmethyl phenyl CO 1047phenylmethyl H proline side-chain phenyl CO 1048 phenylmethyl H1,1-ethano¹ phenyl CO 1049 B phenylmethyl H asparagine phenyl COside-chain 1050 4-(N′-phenylurea)phenylmethyl H isobutyl 1,3-benzo- COdioxol-5-yl 1051 4-(N′-phenylurea)phenyl H isobutyl phenyl CO 10522-[4-(N′-phenylurea)phenyl]- H isobutyl phenyl CO ethyl 10534-(N′-phenylurea)phenylmethyl methyl isobutyl phenyl CO 10543-(N′-phenylurea)phenylmethyl H isobutyl phenyl CO 10554-(N′-phenylurea)phenylmethyl methyl isobutyl 1,3-benzo- CO dioxol-5-yl1056 3-methoxy-4-(N′-phenylurea)- H isobutyl 1,3-benzo- CO phenylmethyldioxol-5-yl 1057 3-hydroxy-4-(N′-phenylurea)- H isobutyl 1,3-benzo- COphenylmethyl dioxol-5-yl 1058 3-methyl-4-(N′-phenylurea)- H isobutyl1,3-benzo- CO phenylmethyl dioxol-5-yl 10604-(N′-phenylurea)phenylmethyl H isobutyl phenyl CO 10634-(N′-phenylurea)phenylmethyl H isobutyl benzyl CO 10644-(N′-methylurea)phenylmethyl H isobutyl 1,3-benzo- CO dioxol-5-yl 10654-(N′-isopropylurea)- H isobutyl 1,3-benzo- CO phenylmethyl dioxol-5-yl1066 4-(N′-phenylurea)phenylmethyl H isobutyl 1,3-benzo- CO dioxol-5-yl1067 4-(N′-p-toluylurea)- H isobutyl 1,3-benzo- CO phenylmethyldioxol-5-yl 1068 4-(N′-cyclohexylurea)- H isobutyl 1,3-benzo- COphenylmethyl dioxol-5-yl 1069 4-(N′-phenylurea)phenylmethyl H isobutyl2-methoxy CO phenyl 1070 4-hydroxyphenylmethyl H isobutyl 2-methoxy COphenyl 1072 4-(N′-phenylurea)phenylmethyl H isobutyl 3-methoxy CO phenyl1073 4-(benzyloxycarbonylamino)- H isobutyl 1,3-benzo- CO phenylmethyldioxol-5-yl 1074 4-(phenylsulfonamido) H isobutyl 1,3-benzo- COphenyl-methyl dioxol-5-yl 1075 4-(benzamido)phenylmethyl H isobutyl1,3-benzo- CO dioxol-5-yl 1076 4-(N′-t-butylurea)phenylmethyl H isobutyl1,3-benzo- CO dioxol-5-yl 1077 4-(N′-ethylurea)phenylmethyl H isobutyl1,3-benzo- CO dioxol-5-yl 1078 4-(N′-(3-methoxyphenyl)urea)- H isobutyl1,3-benzo- CO phenylmethyl dioxol-5-yl 10794-(N′-(2-methoxyphenyl)urea)- H isobutyl 1,3-benzo- CO phenylmethyldioxol-5-yl 1080 4-(N′-(3-pyridyl)urea)- H isobutyl 1,3-benzo- COphenylmethyl dioxol-5-yl 1081 phenylmethyl H isobutyl phenyl CO 10823-phenylpropyl H isobutyl phenyl CO 1083 methyl H isobutyl phenyl CO1084 2-(4-hydroxyphenyl)ethyl H isobutyl phenyl CO 1085 benzyloxy Hisobutyl phenyl CO 1086 N-phenylamino H isobutyl phenyl CO 10872-(4-hydroxyphenyl)ethyl methyl isobutyl phenyl CO 10884-(N′-phenylurea)phenylmethyl H isobutyl 4-methoxy CO phenyl 10894-(N′-phenylurea)phenylmethyl H 2-(methylthio)- 4-methoxy CO ethylphenyl 1090 4-(N′-phenylurea)phenylmethyl H isobutyl 1,3-benzo- COdioxol-5-yl 1091 4-hydroxyphenylmethyl H isobutyl phenyl CO 10924-methoxyphenylmethyl H isobutyl phenyl CO 1093 4-nitrophenytmethyl Hisobutyl phenyl CO 1094 n-hexyl H isobutyl phenyl CO 10962-hydroxyphenylmethyl H isobutyl phenyl CO 1097 3-hydroxyphenylmethyl Hisobutyl phenyl CO 1098 3,4-dihydroxyphenylmethyl H isobutyl phenyl CO1099 2,2-diphenylethyl H isobutyl phenyl CO 1100 2-bromo-4-hydroxy-5- Hisobutyl phenyl CO methoxyphenylmethyl 1101 4-(benzyloxycarbonylamino)-H isobutyl phenyl CO phenylmethyl 1102 2-(N′-phenylurea)phenylmethyl Hisobutyl phenyl CO 1103 4-aminophenylmethyl H isobutyl phenyl CO 11044-(phenylsulfonamido)phenyl- H isobutyl phenyl CO methyl 11054-(benzamido)phenylmethyl H isobutyl phenyl CO 11065-(N′-phenylurea)pentyl H isobutyl phenyl CO 11075-(N′-t-butylurea)pentyl H isobutyl phenyl CO 1108 4-nitrophenylamino Hisobutyl phenyl CO 1109 4-aminophenylamino H isobutyl phenyl 11104-(N′-phenylurea)phenylamino H isobutyl phenyl 11113,5-dimethoxy-4-hydroxy- H isobutyl phenyl phenylmethyl 11124-hydroxy-3-nitrophenylmethyl H isobutyl phenyl 1113 3-nitrophenylmethylH isobutyl phenyl 1114 phenylmethyl methyl isobutyl phenyl CO 1115phenylmethyl H isobutyl 4-chloro CO phenyl 1116 phenylmethyl H1-hydroxy-ethyl phenyl CO 1117 phenylmethyl H 1-methoxy-ethyl phenyl CO1119 phenylmethyl H methyl phenyl CO 1120 phenylmethyl methyl methylphenyl CO 1122 phenylmethyl H 4-methoxy- phenyl CO phenylmethyl 1123phenylmethyl H 2-phenylethyl phenyl CO 1124 phenylmethyl H 4-benzyloxy-phenyl CO phenylmethyl 1125 phenylmethyl H 4-hydroxy- phenyl COphenylmethyl 1126 phenylmethyl H benzyloxy- phenyl CO methyl 1127phenylmethyl H benzylthio- phenyl CO methyl 11284-(N′-phenylurea)phenylmethyl H isobutyl 1,3-benzo- CO dioxol-5-yl 11294-(N′-phenylurea)phenylmethyl H benzyl 1,3-benzo- CO dioxol-5-yl 11304-(N′-phenylurea)phenylmethyl H benzyl phenyl CO 11314-(N′-phenylurea)phenylmethyl H sec-butyl phenyl CO 11324-(N′-phenylurea)phenylmethyl H 4-(benzyloxy- phenyl COcarbonylamino)butyl 1133 4-(N′-phenylurea)phenylmethyl H sec-butyl1,3-benzo- CO dioxol-5-yl 1134 4-(N′-phenylurea)phenylmethyl H t-butoxy-phenyl CO carbonylamino- methyl 1135 4-(N′-phenylurea)phenylmethyl H2-(methylthio)- phenyl CO ethyl 1136 4-(N-phenylurea)phenylmethyl H2-benzylthio- phenyl CO methyl 1137 phenylmethyl H isobutyl 2-nitro COphenyl 1138 4-(N′-phenylurea)phenylmethyl H aminomethyl phenyl CO 11394-(N′-phenylurea)phenylmethyl H 4-amino-butyl phenyl CO 1140phenylcarbonyl H isobutyl phenyl CH₂ 1141 phenylcarbonyl phenacylisobutyl phenyl CH₂ 1142 2,3-benzocyclobutyl H isobutyl phenyl CO 11434-hydroxyphenylmethyl H isobutyl benzyl CO 1144 4-hydroxyphenylmethyl Hisobutyl phenyl CO 1145 4-(t-butoxycarbonylamino)- H isobutyl phenyl COphenylmethyl 1146 4-hydroxyphenylmethyl H isobutyl 3-methoxy CO phenyl1147 4-acetamidophenylmethyl H isobutyl phenyl CO 11484-hydroxyphenylmethyl H isobutyl 3-pyridyl CO 1149 2-quinolinyl Hisobutyl phenyl CO 1150 2-phenylethyl H isobutyl phenyl CO 11522,2-dimethylpropyl H isobutyl phenyl CO 1153 benzyloxy H isobutyl3-pyridyl CO 1154 t-butylamino H isobutyl phenyl CO 1155 phenylmethyl Ht-butyl phenyl CO 1156 methyl H t-butyl phenyl CO 1157 phenylmethyl Hisobutyl benzyl CO 1158 phenylmethyl H isobutyl 1,3-benzo- COdioxol-5-yl 1159 phenylmethyl H isobutyl 2-methoxy CO phenyl 1160phenylmethyl H isobutyl 3-methoxy CO phenyl 1162 benzyloxy H isobutylmethyl CO 1163 4-(N′-phenylurea)phenylmethyl H 2-(methylthio)-1,3-benzo- CO ethyl dioxol-5-yl 1164 phenylmethyl H 2-(methylthio)-1,3-benzo- CO ethyl dioxol-5-yl 1168 4-(N′-(m-toluyl)urea)- H isobutyl1,3-benzo- CO phenylmethyl dioxol-5-yl 1169 4-(N′-benzylurea)- Hisobutyl 1,3-benzo- CO phenylmethyl dioxol-5-yl 11704-(N′-phenylurea)phenylmethyl H morpholino-N- 1,3-benzo- COcarbonylmethyl dioxol-5-yl 1173 4-hydroxyphenylmethyl H isobutyl4-methoxy CO phenyl 1174 4-hydroxyphenylmethyl H 2-(methylthio)-4-methoxy CO ethyl phenyl 1175 phenylmethyl H 2-(methylthio)- 4-methoxyCO ethyl phenyl 1176 4-(N′-phenylurea)phenylmethyl H thiomorpholino-N-1,3-benzo- CO carbonylmethyl dioxol-5-yl 11774-(N′-phenylurea)phenylmethyl H N,N-(methylprop 1,3-benzo- COargyl)amino dioxol-5-yl carbonyl-methyl 1178 phenylmethyl H isobutyl4-methoxy CO phenyl 1179 4-(N′-(o-toluyl)urea)- H isobutyl 1,3-benzo- COphenylmethyl dioxol-5-yl 1180 4-(N′-(2-thiazolyl)urea)- H isobutyl4-methoxy CO phenylmethyl phenyl 1181 4-(N′-(3-chlorophenyl)urea)- Hisobutyl 1,3-benzo- CO phenylmethyl dioxol-5-yl 11824-(N′-(4-pyridyl)urea)- H isobutyl 1,3-benzo- CO phenylmethyldioxol-5-yl 1185 4-(N′-(2-chlorophenyl)urea)- H isobutyl 1,3-benzo- COphenylmethyl dioxol-5-yl 1186 4-(N′-phenylurea)phenylmethyl H isobutylisobutylamino- CO carbonyl 1187 3-(N-phenylurea)propyl H isobutyl phenylCO 1188 1-phenylcyclopropyl H isobutyl 1,3-benzo- CO dioxol-5-yl 11891-indanyl H isobutyl phenyl CO 1190 4-(N′-(o-toluyl)urea)- H isobutyl4-methoxy CO phenylmethyl phenyl 1191 4-(N′-phenylurea)phenylmethyl H2-(N-morpholino)- 1,3-benzo- CO ethyl dioxol-5-yl 11924-(N′-(2-methoxyphenyl)urea)- H isobutyl 4-methoxy CO phenylmethylphenyl 1193 4-(N′-phenylurea)phenylmethyl methyl isobutyl 4-methoxy COphenyl 1194 4-(N′-(2-pyridyl)urea)- H isobutyl 4-methoxy CO phenylmethylphenyl 1195 4-(N′-phenylurea)phenylmethyl H isobutyl 3,4-difluoro- COphenyl 1196 4-(N′-phenylurea)phenylmethyl H isobutyl 3,4-dimeth- COoxy-phenyl 1197 4-(N′-(o-toluyl)urea)- H isobutyl phenyl CO phenylmethyl1198 4-(morpholinocarbonylamino)- H isobutyl 1,3-benzo- CO phenylmethyldioxol-5-yl 1199 4-(N′-phenylurea)phenylmethyl H 2-methyl- 4-methoxy COsulfinylethyl phenyl 1200 4-(N′-(2-ethylphenyl)urea)- H isobutyl1,3-benzo- CO phenylmethyl dioxol-5-yl 1201 4-(N′-(2-nitrophenyl)urea)-H isobutyl 1,3-benzo- CO phenylmethyl dioxol-5-yl 12064-(N′-(2-isopropylphenyl)urea)- H isobutyl 1,3-benzo- CO phenylmethyldioxol-5-yl 1207 4-(N′-(2-isopropylphenyl)urea)- H isobutyl 4-methoxy COphenylmethyl phenyl 1208 4-(N′-(2-ethylphenyl)urea)- H isobutyl4-methoxy CO phenylmethyl phenyl 1209 4-(N′-(2-t-butylphenyl)urea)- Hisobutyl 1,3-benzo- CO phenylmethyl dioxol-5-yl 12104-(N′-(o-toluyl)urea)- H isobutyl 1,3-benzo- CO phenylmethyl dioxol-5-yl1212 4-(N′-(o-toluyl)urea)- H isobutyl 3,4-dimethoxy CO phenylmethylphenyl 1214 4-(N′-phenylurea)phenylmethyl H N,N-dimethyl 1,3-benzo- COamino- dioxol-5-yl carbonylmethyl 1215 4-(N′-phenylurea)phenylmethyl H2-(N,N-dimethyl- 1,3-benzo- amino)-ethyl dioxol-5-yl 12164-(N′-phenylurea)phenylmethyl H 2-(morpholino-N- 1,3-benzo- COcarbonyl)-ethyl dioxol-5-yl 1217 4-(N′-(o-toluyl)urea)- H 4-(benzyloxy-3,4-dimethoxy CO phenylmethyl carbonylamino)- phenyl butyl 12184-(N′-(2-pyridyl)urea)- H isobutyl 3,4-dimethoxy CO phenylmethyl phenyl1219 4-(N′-(3-pyridyl)urea)- H isobutyl 3,4-dimethoxy CO phenylmethylphenyl 1220 4-(N′-(2-methyl-3-pyridyl) H isobutyl 4-methoxy COurea)-phenylmethyl phenyl 1221 3-methoxy-4-(N′-(o-toluyl)- H isobutyl1,3-benzo- CO urea)phenylmethyl dioxol-5-yl 12224-(N′-(2-chlorophenyl)urea)- H isobutyl 1,3-benzo- CO3-methoxyphenylmethyl dioxol-5-yl 1223 4-(phenylaminocarbonylamino- Hisobutyl 1,3-benzo- CO methyl)-phenyl dioxol-5-yl 12244-(N′-(o-toluyl)urea)- H 2-(methylthio)- 3,4-dimethoxy CO phenylmethylethyl phenyl 1225 4-(N′-(o-toluyl)urea)- H 4-(benzyloxy- 1,3-benzo- COphenylmethyl carbonylamino)- dioxol-5-yl butyl 12274-(N′-(o-toluyl)urea)- H methylthiomethyl 1,3-benzo- CO phenylmethyldioxol-5-yl 1238 4-(N′-(o-toluyl)urea)- H 2-(methylthio)- 4-methoxy COphenylmethyl ethyl phenyl 1245 4-(N′-(o-toluyl)urea)- H2-(methyl-sulfonyl)- 1,3-benzo- CO phenylmethyl ethyl dioxol-5-yl 12464-(N′-(o-toluyl)urea)- H 3-(hyrdoxypropy- 1,3-benzo- CO phenylmethylthio)-methyl dioxol-5-yl 1248 4-(N′-(o-toluyl)urea)- H isobutyl4-fluorophenyl CO phenylmethyl 1270 4-(N′-(o-toluyl)urea)- H4-acetylamino- 1,3-benzo- CO phenylmethyl butyl dioxol-5-yl 12724-(N′-(2-methylphenyl) H 4-(methoxy 1,3-benzo- CO urea) phenylmethylcarbonyl amino) dioxol-5-yl butyl 1282 4-(N′-(o-toluyl)urea)-pyrid-5- Hisobutyl 1,3-benzo- CO yllmethyl dioxol-5-yl 1294 4-(N′-(o-toluyl)urea)-H 4-(methylsulfonyl- 1,3-benzo- CO phenylmethyl amino)-butyl dioxol-5-yl1311 4-(N′-(3-methyl-2-pyridyl) H 4-(methoxy 3,4-dimethoxy CO urea)phenylmethyl carbonyl amino) phenyl butyl 1319 4-(indolylcarbonyl Hisobutyl 1,3-benzo- CO amino)phenylmethyl dioxol-5-yl 13214-(N′-(o-toluyl)urea)- H isobutyl 4- CO phenylmethyl carboxyphenyl 13274-(1-indolecarboxylamino)- H isobutyl 1,3-benzo- CO phenylmethyldioxol-5-yl 1336 6-methoxy-5-(N′-(o-toluyl) H isobutyl 1,3-benzo- COurea)-2-pyridylmethyl dioxol-5-yl 1345 4-(N′-(o-toluyl)urea)- Hdimethylamino 1,3-benzo- CO phenylmethyl ethylthiomethyl dioxol-5-yl1347 4-(N′-2-pyridyl) H 2-(methylthio)- 3,4-dimethoxy- COurea)phenylmethyl ethyl phenyl 1358 4-(N′-phenylthiourea) H isobutyl1,3-benzo- CO phenylmethyl dioxol-5-yl 1360 4-(N′-(o-toluyl)urea)- Hisobutyl 2,3-dihydro- CO phenylmethyl benzofuran-5-yl 13614-(N′-(o-toluyl)urea)- H methylthio 4-carbometh- CO phenylmethyl ethyloxy phenyl 1380 4-(N′-phenyl-N″-methyl- H isobutyl 1,3-benzo- COguanidino)-phenylmethyl dioxol-5-yl 1382 4-(N′-(o-toluyl)urea)- H4-(methylsulfonyl- 4-carbometh- CO phenylmethyl amino)-butyl oxy-phenyl1388 4-(phenylurea) H isobutyl 4-carbometh- CO phenylmethyl oxy-phenyl1390 4-(1,3-imidazol-2-ylamino)- H isobutyl 1,3-benzo- CO phenylmethyldioxol-5-yl 1393 4-(N′-(2-pyridyl) H 2-(methylthio)- 1,3-benzo- CO urea)phenylmethyl ethyl dioxol-5-yl 1396 4-(1,3-benzoxazol-2-ylamino)- Hisobutyl 1,3-benzo- CO phenylmethyl dioxol-5-yl 14004-(N′-(2-methylphenyl)urea)- H isobutyl phenylethyl CO phenylmethyl 14294-(N′-(3-methyl-2-pyridyl) H 2-(methylthio)- 1,3-benzo- CO urea)phenylmethyl ethyl dioxol-5-yl 1444 4-(2-benzoxazolinonyl H isobutyl1,3-benzo- CO carbonylamino)phenyl- dioxol-5-yl methyl 14744-(2-pyrrolylcarbonylamino) H isobutyl 1,3-benzo- CO phenylmethyldioxol-5-yl 1475 4-(N′-allylurea)phenyl- H isobutyl 1,3-benzo- CO methyldioxol-5-yl 1490 4-(N′-(2-methylphenyl) H isobutyl ethynyl COurea)phenylmethyl 1515 4-(N′-(2-methylphenyl) H isobutyl allyl COurea)phenylmethyl 1525 4-(N′-(2-fluorophenyl) H isobutyl 3,4-diemthoxy-CO urea)phenylmethyl phenyl 1526 4-(4-fluorophenylurea) H isobutyl3,4-dimethoxy CO phenylmethyl phenyl 1536 4-(N′-(2-methylphenyl) Hisobutyl methyl CO urea)phenylmethyl 1594 4-(N′-2-methylpenylurea)- Hisobutyl H CO phenylmethyl 1648 4-(2-indoylycarbonylamino) H isobutyl HCO phenylmethyl 1655 4-(3-indolylcarbonylamino) H isobutyl 1,3-benzo- COphenylmethyl dioxol-5-yl 1721 4-(N′-(2-methylphenyl) H isobutylmorpholino- CO urea)phenylmethyl methyl 1725 3-methoxy-4-(N′-phenylurea) H 2-(methylthio)- 1,3-benzo- CO phenylmethyl ethyl dioxol-5-yl1726 3-methoxy-4-(N′-phenyl urea) H isobutyl 3,4-dimethoxy- COphenylmethyl phenyl 1727 3-methoxy-4-(N′-phenyl urea) H 2-(methylthio)-3,4-dimethoxy- CO phenylmethyl ethyl phenyl 17283-methoxy-4-(N′-2-pyridyl urea) H isobutyl 3,4-dimethoxy- COphenylmethyl phenyl 1729 3-methoxy-4-(N′-3-methyl-2- H isobutyl3,4-dimethoxy- CO pyridyl) urea phenylmethyl phenyl 17303-methoxy-4-(N′-3-methyl-2- H 2-(methylthio)- 3,4-dimethoxy- CO pyridyl)urea phenylmethyl ethyl phenyl 1731 3-methoxy-4-(N′-3-methyl-2- H2-(methylthio)- 1,3-benzo- CO pyridyl) urea phenylmethyl ethyldioxol-5-yl 1732 4-(N′-(3-methyl-2-pyridyl) H 2-(methylthio)-3,4-dimethoxy- CO urea) phenylmethyl ethyl phenyl

The more preferred compounds of formula (I) are: BIO-1006, BIO-1056,BIO-1089, BIO-1179, BIO-1194, BIO-1221, BIO-1224, BIO-1238, BIO-1245,BIO-1246, BIO-1248, BIO-1270, BIO-1282, BIO-1294, BIO-1321, BIO-1336BIO-1382 and BIO-1400. Even more preferred compounds are BIO-1218,BIO-1272, BIO-1311, BIO-1319, BIO-1345, BIO-1347, BIO-1358, BIO-1361,BIO 1388, BIO-1390, BIO-1393, BIO-1396, BIO-1429, BIO-1444, BIO-1474,BIO-1475, BIO-1490, BIO-1515, BIO-1525, BIO-1526, BIO-1536, BIO-1594,BIO-1648, BIO-1655, BIO-1721, BIO-1725, BIO-1726, BIO-1727, BIO-1728,BIO-1729, BIO-1730, BIO-1731, and BIO-1732. Most preferred are BIO-1218,BIO-1272, BIO-1311, BIO-1347, BIO-1393, BIO-1429, BIO-1515, BIO-1725,BIO-1726, BIO-1727, BIO-1728, BIO-1729, BIO-1730, BIO-1731, andBIO-1732.

Compounds of this invention may be synthesized using any conventionaltechnique. Preferably, these compounds are chemically synthesized fromreadily available starting materials, such as α-amino acids. Modular andconvergent methods for the synthesis of these compounds are alsopreferred. In a convergent approach, for example, large sections of thefinal product are brought together in the last stages of the synthesis,rather than by incremental addition of small pieces to a growingmolecular chain.

According to one embodiment, compounds of the present invention may besynthesized in the following manner. A protected chiral amine is addedto an α,β-unsaturated ester to produce a protected β-amino acid ester.Upon suitable deprotection, the β-amino acid ester is coupled to anappropriate activated ester moiety. The coupled product, if suitablyfunctionalized, may be further reacted with yet another activated estermoiety. This material can be further manipulated to give the desiredcompounds of the invention. At each step of the above sequence, theester can be hydrolyzed to the corresponding acid to give anothercompound of the invention.

Alternatively, the activated ester moieties mentioned above can beattached together first, then the resulting compound can be attached tothe β-amino acid ester portion. At this point the final manipulationsand/or necessary deprotection steps can be performed.

Alternatively, under suitable conditions, the desired functionalitiescan be incorporated (protected or unprotected) in one of the activatedester moieties. That piece is then coupled with a β-amino acid ester ora moiety consisting of a β-amino ester previously coupled to anactivated ester. The resulting product can then be subjected to anydeprotection steps, if necessary, to give compounds of the invention.

Alternatively, the chiral β-amino acid esters used in the synthesis ofthe compounds of this invention may be synthesized by well-knowntechniques, such as those described in U.S. Pat. No. 5,344,957, thedisclosure of which is herein incorporated by references.

The compounds of this invention may also be modified by appendingappropriate functionalities to enhance selective biological properties.Such modifications are known in the art and include those which increasebiological penetration into a given biological system (e.g., blood,lymphatic system, central nervous system), increase oral availability,increase solubility to allow administration by injection, altermetabolism and alter rate of excretion.

As used throughout this application, the term “patient” refers tomammals, including humans. And the term “cell” refers to mammaliancells, including human cells.

Once synthesized, the activities and VLA-4 specificities of thecompounds according to this invention may be determined using in vitroand in vivo assays.

For example, the cell adhesion inhibitory activity of these compoundsmay be measured by determining the concentration of inhibitor requiredto block the binding of VLA-4-expressing cells to fibronectin- orCS1-coated plates. In this assay microtiter wells are coated with eitherfibronectin (containing the CS-1 sequence) or CS-1. If CS-1 is used, itmust be conjugated to a carrier protein, such as bovine serum albumin,in order to bind to the wells. Once the wells are coated, varyingconcentrations of the test compound are then added together withappropriately labelled, VLA-4-expressing cells. Alternatively, the testcompound may be added first and allowed to incubate with the coatedwells prior to the addition of the cells. The cells are allowed toincubate in the wells for at least 30 minutes. Following incubation, thewells are emptied and washed. Inhibition of binding is measured byquantitating the fluorescence or radioactivity bound to the plate foreach of the various concentrations of test compound, as well as forcontrols containing no test compound.

VLA-4-expressing cells that may be utilized in this assay include Ramoscells, Jurkat cells, A375 melanoma cells, as well as human peripheralblood lymophocytes (PBLs). The cells used in this assay may befluorescently or radioactively labelled.

A direct binding assay may also be employed to quantitate the inhibitoryactivity of the compounds of this invention. In this assay, a VCAM-IgGfusion protein containing the first two immunoglobin domains of VCAM(D1D2) attached above the hinge region of an IgG1 molecule (“VCAM2D-IgG”), is conjugated to a marker enzyme, such as alkaline phosphatase(“AP”). The synthesis of this VCAM-IgG fusion is described in PCTpublication WO 90/13300, the disclosure of which is herein incorporatedby reference. The conjugation of that fusion to a marker enzyme isachieved by cross-linking methods well-known in the art.

The VCAM-IgG enzyme conjugate is then placed in the wells of a muti-wellfiltration plate, such as that contained in the Millipore MultiscreenAssay System (Millipore Corp., Bedford, Mass.). Varying concentrationsof the test inhibitory compound are then added to the wells followed byaddition of VLA-4-expressing cells. The cells, compound and VCAM-IgGenzyme conjugate are mixed together and allowed to incubate at roomtemperature.

Following incubation, the wells are vacuum drained, leaving behind thecells and any bound VCAM. Quantitation of bound VCAM is determined byadding an appropriate calorimetric substrate for the enzyme conjugatedto VCAM-IgG and determining the amount of reaction product. Decreasedreaction product indicates increased cell adhesion inhibitory activity.

In order to assess the VLA-4 inhibitory specificity of the compounds ofthis invention, assays for other major groups of integrins, i.e., α2 andα3, as well as other α1 integrins, such as VLA-5, VLA-6 and α4β7 areperformed. These assays may be similar to the adhesion inhibition anddirect binding assays described above, substituting the appropriateintegrin-expressing cell and corresponding ligand. For example,polymorphonuclear cells (PMNs) express α2 integrins on their surface andbind to ICAM. α3 integrins are involved in platelet aggregation andinhibition may be measured in a standard platelet aggregation assay.VLA-5 binds specifically to Arg-Gly-Asp sequences, while VLA-6 binds tolaminin. α4β7 is a recently discovered homologue of VLA-4, which alsobinds fibronectin and VCAM. Specificity with respect to α4β7 isdetermined in a binding assay that utilizes the above-describedVCAM-IgG-enzyme marker conjugate and a cell line that expresses α4β7,but not VLA-4, such as RPMI-8866 cells.

Once VLA-4-specific inhibitors are identified, they may be furthercharacterized in in vivo assays. One such assay tests the inhibition ofcontact hypersensitivity in an animal, such as described by P. L.Chisholm et al., “Monoclonal Antibodies to the Integrin α-4 SubunitInhibit the Murine Contact Hypersensitivity Response”, Eur. J. Immunol.,23, pp. 682-688 (1993) and in “Current Protocols in Immunology”, J. E.Coligan, et al., Eds., John Wiley & Sons, New York, 1, pp. 4.2.1-4.2.5(1991), the disclosures of which is herein incorporated by reference. Inthis assay, the skin of the animal is sensitized by exposure to anirritant, such as dinitrofluorobenzene, followed by light physicalirritation, such as scratching the skin lightly with a sharp edge.Following a recovery period, the animals are re-sensitized following thesame procedure. Several days after sensitization, one ear of the animalis exposed to the chemical irritant, while the other ear is treated witha non-irritant control solution. Shortly after treating the ears, theanimals are given various doses of the VLA-4 inhibitor by subcutaneousinjection. In vivo inhibition of cell adhesion-associated inflammationis assessed by measuring the ear swelling response of the animal in thetreated versus untreated ear. Swellling is measured using calipers orother suitable instrument to measure ear thickness. In this manner, onemay identify those inhibitors of this invention which are best suitedfor inhibiting inflammation.

Another in vivo assay that may be employed to test the inhibitors ofthis invention is the sheep asthma assay. This assay is performedessentially as described in W. M. Abraham et al., “α-Integrins MediateAntigeninduced Late Bronchial Responses and Prolonged AirwayHyperresponsiveness in Sheep, ” J. Clin. Invest., 93, pp. 776-87 (1994),the disclosure of which is herein incorporated by reference. This assaymeasures inhibition of Ascaris antigen-induced late phase airwayresponses and airway hyperresponsiveness in asthmatic sheep.

The compounds of the present invention may be used in the form ofpharmaceutically acceptable salts derived from inorganic or organicacids and bases. Included among such acid salts are the following:acetate, adipate, alginate, aspartate, benzoate, benzenesulfonate,bisulfate, butyrate, citrate, camphorate, camphorsulfonate,cyclopentanepropionate, digluconate, dodecylsulfate, ethanesulfonate,fumarate, glucoheptanoate, glycerophosphate, hemisulfate, heptanoate,hexanoate, hydrochloride, hydrobromide, hydroiodide,2-hydroxyethanesulfonate, lactate, maleate, methanesulfonate,2-naphthalenesulfonate, nicotinate, oxalate, pamoate, pectinate,persulfate, 3-phenyl-propionate, picrate, pivalate, propionate,succinate, tartrate, thiocyanate, tosylate and undecanoate. Base saltsinclude ammonium salts, alkali metal salts, such as sodium and potassiumsalts, alkaline earth metal salts, such as calcium and magnesium salts,salts with organic bases, such as dicyclohexylamine salts,N-methyl-D-glucamine, and salts with amino acids such as arginine,lysine, and so forth. Also, the basic nitrogen-containing groups can bequaternized with such agents as lower alkyl halides, such as methyl,ethyl, propyl, and butyl chloride, bromides and iodides; dialkylsulfates, such as dimethyl, diethyl, dibutyl and diamyl sulfates, longchain halides such as decyl, lauryl, myristyl and stearyl chlorides,bromides and iodides, aralkyl halides, such as benzyl and phenethylbromides and others. Water or oil-soluble or dispersible products arethereby obtained.

The compounds of the present invention may be formulated intopharmaceutical compositions that may be administered orally,parenterally, by inhalation spray, topically, rectally, nasally,buccally, vaginally or via an implanted reservoir. The term “parenteral”as used herein includes subcutaneous, intravenous, intramuscular,intra-articular, intra-synovial, intrasternal, intrathecal,intrahepatic, intralesional and intracranial injection or infusiontechniques.

The pharmaceutical compositions of this invention comprise any of thecompounds of the present invention, or pharmaceutically acceptable saltsthereof, together with any pharmaceutically acceptable carrier. The term“carrier” as used herein includes acceptable adjuvants and vehicles.Pharmaceutically acceptable carriers that may be used in thepharmaceutical compositions of this invention include, but are notlimited to, ion exchangers, alumina, aluminum stearate, lecithin, serumproteins, such as human serum albumin, buffer substances such asphosphates, glycine, sorbic acid, potassium sorbate, partial glyceridemixtures of saturated vegetable fatty acids, water, salts orelectrolytes, such as protamine sulfate, disodium hydrogen phosphate,potassium hydrogen phosphate, sodium chloride, zinc salts, colloidalsilica, magnesium trisilicate, polyvinyl pyrrolidone, cellulose-basedsubstances, polyethylene glycol, sodium carboxymethylcellulose,polyacrylates, waxes, polyethylene-polyoxypropylene-block polymers,polyethylene glycol and wool fat.

According to this invention, the pharmaceutical compositions may be inthe form of a sterile injectable preparation, for example a sterileinjectable aqueous or oleaginous suspension. This suspension may beformulated according to techniques known in the art using suitabledispersing or wetting agents and suspending agents. The sterileinjectable preparation may also be a sterile injectable solution orsuspension in a non-toxic parenterally-acceptable diluent or solvent,for example as a solution in 1,3-butanediol. Among the acceptablevehicles and solvents that may be employed are water, Ringer's solutionand isotonic sodium chloride solution. In addition, sterile, fixed oilsare conventionally employed as a solvent or suspending medium. For thispurpose, any bland fixed oil may be employed including synthetic mono-or di-glycerides. Fatty acids, such as oleic acid and its glyceridederivatives are useful in the preparation of injectables, as do naturalpharmaceutically-acceptable oils, such as olive oil or castor oil,especially in their polyoxyethylated versions. These oil solutions orsuspensions may also contain a long-chain alcohol diluent or dispersant,such as Ph. Helv or similar alcohol.

The pharmaceutical compositions of this invention may be orallyadministered in any orally acceptable dosage form including, but notlimited to, capsules, tablets, aqueous suspensions or solutions. In thecase of tablets for oral use, carriers which are commonly used includelactose and corn starch. Lubricating agents, such as magnesium stearate,are also typically added. For oral administration in a capsule form,useful diluents include lactose and dried corn starch. When aqueoussuspensions are required for oral use, the active ingredient is combinedwith emulsifying and suspending agents. If desired, certain sweetening,flavoring or coloring agents may also be added.

Alternatively, the pharmaceutical compositions of this invention may beadministered in the form of suppositories for rectal administration.These can be prepared by mixing the agent with a suitable non-irritatingexcipient which is solid at room temperature but liquid at the rectaltemperature and therefore will melt in the rectum to release the drug.Such materials include cocoa butter, beeswax and polyethylene glycols.

The pharmaceutical compositions of this invention may also beadministered topically, especially when the target of treatment includesareas or organs readily accessible by topical application, includingdiseases of the eye, the skin, or the lower intestinal tract. Suitabletopical formulations are readily prepared for each of these areas ororgans.

Topical application for the lower intestinal tract can be effected in arectal suppository formulation (see above) or in a suitable enemaformulation. Topically-transdermal patches may also be used.

For topical applications, the pharmaceutical compositions may beformulated in a suitable ointment containing the active componentsuspended or dissolved in one or more carriers. Carriers for topicaladministration of the compounds of this invention include, but are notlimited to, mineral oil, liquid petrolatum, white petrolatum, propyleneglycol, polyoxyethylene, polyoxypropylene compound, emulsifying wax andwater. Alternatively, the pharmaceutical compositions can be formulatedin a suitable lotion or cream containing the active components suspendedor dissolved in one or more pharmaceutically acceptable carriers.Suitable carriers include, but are not limited to, mineral oil, sorbitanmonostearate, polysorbate 60, cetyl esters wax, cetearyl alcohol,2-octyldodecanol, benzyl alcohol and water.

For ophthalmic use, the pharmaceutical compositions may be formulated asmicronized suspensions in isotonic, pH adjusted sterile saline, or,preferably, as solutions in isotonic, pH adjusted sterile saline, eitherwith our without a preservative such as benzylalkonium chloride.Alternatively, for ophthalmic uses, the pharmaceutical compositions maybe formulated in an ointment such as petrolatum.

The pharmaceutical compositions of this invention may also beadministered by nasal aerosol or inhalation through the use of anebulizer, a dry powder inhaler or a metered dose inhaler. Suchcompositions are prepared according to techniques well-known in the artof pharmaceutical formulation and may be prepared as solutions insaline, employing benzyl alcohol or other suitable preservatives,absorption promoters to enhance bioavailability, fluorocarbons, and/orother conventional solubilizing or dispersing agents.

The amount of active ingredient that may be combined with the carriermaterials to produce a single dosage form will vary depending upon thehost treated, and the particular mode of administration. It should beunderstood, however, that a specific dosage and treatment regimen forany particular patient will depend upon a variety of factors, includingthe activity of the specific compound employed, the age, body weight,general health, sex, diet, time of administration, rate of excretion,drug combination, and the judgment of the treating physician and theseverity of the particular disease being treated. The amount of activeingredient may also depend upon the therapeutic or prophylactic agent,if any, with which the ingredient is co-administered.

The dosage and dose rate of the compounds of this invention effective toprevent, suppress or inhibit cell adhesion will depend on a variety offactors, such as the nature of the inhibitor, the size of the patient,the goal of the treatment, the nature of the pathology to be treated,the specific pharmaceutical composition used, and the judgment of thetreating physician. Dosage levels of between about 0.001 and about 100mg/kg body weight per day, preferably between about 0.1 and about 10mg/kg body weight per day of the active ingredient compound are useful.

According to another embodiment compositions containing a compound ofthis invention may also comprise an additional agent selected from thegroup consisting of corticosteroids, bronchodilators, antiasthmatics(mast cell stabilizers), anti-inflammatories, antirheumatics,immunosuppressants, antimetabolites, immunonodulators, antipsoriaticsand antidiabetics. Specific compounds within each of these classes maybe selected from any of those listed under the appropriate groupheadings in “Comprehensive Medicinal Chemistry,” Pergamon Press, Oxford,England, pp. 970-986 (1990), the disclosure of which is hereinincorporated by reference. Also included within this group are compoundssuch as theophylline, sulfasalazine and aminosalicylates(antiinflammatories); cyclosporin, FK-506, and rapamycin(immunosuppressants); cyclophosphamide and methotrexate(antimetabolites); and interferons (immunomodulators).

According to other embodiments, the invention provides methods forpreventing, inhibiting or suppressing cell adhesion-associatedinflammation and cell adhesion-associated immune or autoimmuneresponses. VLA4-associated cell adhesion plays a central role in avariety of inflammation, immune and autoimmune diseases. Thus,inhibition of cell adhesion by the compounds of this invention may beutilized in methods of treating or preventing inflammatory, immune andautoimmune diseases. Preferaby the diseases to be treated with themethods of this invention are selected from asthma, arthritis,psoriasis, transplantation rejection, multiple sclerosis, diabetes andinflammatory bowel disease.

These methods may employ the compounds of this invention in amonotherapy or in combination with an anti-inflammatory orimmunosuppressive agent. Such combination therapies includeadministration of the agents in a single dosage form or in multipledosage forms administered at the same time or at different times.

In order that this invention may be more fully understood, the followingexamples are set forth. These examples are for the purpose ofillustration only and are not to be construed as limiting the scope ofthe invention in any way.

Procedure A—Synthesis of Cinnamate Esters

Method A: To a cinnamic acid or substituted cinnamic acid (1.0 mmol) inCH₂Cl₂ (10 ml) was added (COCl)₂ (1.5 mmol) slowly. The reaction mixturewas stirred at r.t. for 4 h and the solvent was removed in vacuo toafford the acid chloride. Methanol or t-butyl alcohol (5 ml) was addedto quantitatively provide the methyl or t-butyl ester after removal ofthe solvents.

Method B: To an appropriate aldehyde (1.0 mmol) in THF (10 ml) was addedt-butoxycarbonyl methylene triphenylphosphorane (1.0 mmol, Aldrich) andthe resulting mixture was stirred at room temperature for 16 h. Thereaction mixture was diluted with petroleum ether (10 ml) and wasfiltered through a pad of celite. The filtrate was collected andconcentrated in vacuo to afford the desired product.

Method A; Yield: 95%; (CDCl₃′ 300 MHz, ppm): 7.57 (d, 1H, J=16 Hz), 7.47(m, 2H), 7.34 (m, 3H), 6.35 (d, 1H, J=16 Hz), 1.52 (s, 9H);

Method B; Yield: 90%; (CDCl₃′ 300 MHz, ppm): 7.48 (d, 1H), 7.28-7.18 (m,5H), 5.69 (d, 2H), 3.44 (d, 2H), 1.42 (s, 9H);

Method A; Yield: 94%; (CDCl₃′ 300 MHz, ppm): 7.95 (d, 1H, J=16 Hz), 7.49(d, 1H), 7.42 (t, 1H), 6.94 (dd, 2H), 6.51 (d, 2H), J=16 Hz), 3.86 (s,3H), 3.76 (s, 3H);

Method A; Yield: 92%; (CDCl₃′ 300 MHz, ppm): 7.52 (d, 1H, J=15.9 Hz),7.28 (t, 1H), 7.09 (d, 1H), 7.02 (br, s, 1H), 6.89 (d, 1H), 6.34 (d, 1H,J=15.9 Hz) 3.82 (s, 3H), 1.54 (s, 9H);

Method A: Yield: 98%; (CDCl₃′ 300 MHz, ppm): 7.64 (d, 1H, J=16 Hz), 7.29(t, 1H), 7.10 (d, 1H), 7.06 (br, s, 1H), 6.94 (d, 1H, J=16 Hz), 3.82 (s,3H), 3.80 (s, 3H);

Method B; Yield: 88%; (CDCl₃′ 300 MHz, ppm): 8.62 (br,s, 1H), 8.51 (m,1H), 7.72 (d, 1H), 7.48 (d, 1H, J=15.9 Hz), 7.22 (m, 1H), 6.36 (d, 1H,J=15.9 Hz), 1.49 (s, 9H);

Method B; Yield: 90%; (CDCl₃′ 300 MHz, ppm): 8.60 (br, s, 1H), 7.66 (t,1H), 7.55 (d, 1H, J=15.9 Hz), 7.36 (d, 1H), 7.21 (m, 1H), 6.78 (d, 1H,J=15.9 Hz), 1.52 (s, 9H);

Method A; Yield: 91%; (CDCl₃′ 300 MHz, ppm): 7.52 (d, 1H, J=15.9 Hz),7.44 (d, 1H, J=8.0 Hz), 6.85 (d, 1H, J=8.0 Hz), 6.21 (d, 1H, J=15.9 Hz),3.81 (s, 3H), 1.52 (s, 9H);

Method A; Yield: 90%; (CDCl₃′ 300 MHz, ppm): 7.61 (d, 1H, J=16 Hz), 7.42(d, 2H, J=7.9 Hz), 6.86 (d, 1H, J=7.9 Hz), 6.28 (d, 1H, J=16 Hz), 3.78(s, 3H), 3.74 (s, 3H);

Method B; Yield: 91%; (CDCl₃′ 300 MHz, ppm): 7.56 (d, 1H, J=16 Hz), 7.46(t, 2H), 7.02 (t, 2H), 6.26 (d, 2H, J=16 Hz), 1.54 (s, 9H)

Method A; Yield: 89%; (CDCl₃′ 300 MHz, ppm): 7.47 (d, 1H, J=15.9 Hz),7.01 (d, 1H, J=8.3 Hz) 6.98 (br, s, 1H), 6.78 (d, 1H, J=8.3 Hz), 3.84(s, 6H), 1.48 (s, 9H)

Method A; Yield: 91%; (CDCl₃′ 300 MHz, ppm): 7.61 (d, 1H, J=15.9 Hz,7.07 (d, 1H, J=8.3 Hz), 7.02 (br, s, 1H), 6.83 (d, 1H, J=8.3 Hz), 6.28(d, 1H, J=15.9 Hz), 3.88 (s, 3H), 3.76 (s, 3H);

Method A; Yield: 92%; (CDCl₃′ 300 MHz, ppm):7.46 (d, 1H, J=16.1 Hz),6.99 (s, 1H), 6.97 (d, 1H), 6.76 (d, 1H), 6.18 (d, 1H, J=16.1 Hz), 5.96(s, 2H), 1.50 (s, 9H);

Method A; Yield: 88%; (CDCl₃′ 300 MHz, ppm): 7.55 (d, 1H, J=15.9 Hz),6.98-6.75 (m, 2H), 6.22 (d, 1H, J=15.9 Hz), 5.96 (s, 2H), 3.75 (s, 3H);

Method B; Yield: 89%; (CDCl₃′ 300 MHz, ppm): 7.45 (d,1H, J=15.8 Hz),6.99 (s, 1H), 6.98 (d, 1H), 6.80 (d, 1H), 6.18 (d, 1H, J=15.8 Hz), 4.21(br,s, 4H), 1.49 s, 9H);

Method B; Yield: 88%.

Method B; Yield: 93%; ¹HNMR(CDCl₃): δ 8.00 (2H, d, J=5.5 Hz), 7.53 (2H,d, J=5.5 Hz), 7.58 (1H,d,J=10,7 Hz), 6.42(1H,d,J=10.7 Hz), 3.90(3H, s),1.51(9H, s).

Procedure B—Synthesis of β-Amino Acids

A 2 L round bottom flask, equipped with a magnetic stir bar, was chargedwith 1000 mL of MeOH and the flask tared with its contents. AnhydrousHCl (11 g, 0.29 mol) was bubbled in from a cylinder. To this solutionwas added a cinnamic acid (0.29 mol) neat in one portion. The resultingmixture was heated at reflux until the reaction was judged complete byTLC analysis. The reaction was cooled to RT, then refrigeratedovernight. The crystalline product was collected by suction filtrationon a medium frit and the cake washed with cold MeOH. The solid was driedon the filter to give a white or nearly white product.

Precursor to β-3: Yield: 94%; TLC (3:1 hexane/EtOAc; UV): R_(f)=0.48;mp=134-136° C.; ¹H NMR (CDCl₃, 300 MHz): 7.58 (d, 1H, J=15.9 Hz),7.00-6.97 (m, 3H), 6.79 (d, 1H, J=7.9 Hz), 6.24 (d, 1H, J=15.9 Hz), 5.98(s, 2H), 3.77 (s, 3H); MS (FAB): 206.

Precursor to β-5: Yield: 84%; TLC (3:1 hexane/EtOAc; UV): R_(f) =0.48;mp=89-91° C.; ¹H NMR (CDCl₃, 300 MHz): 7.63 (d, 1H, J=15.9 Hz), 7.46 (d,2H, J=8.7 Hz), 6.89 (d, 2H, J=8.7 Hz), 6.29 (d, 1H, J=15.9 Hz), 3.82 (s,3H), 3.77 (s, 3H); MS (FAB): 192.

Michael addition of (R)-(+)-N-benzyl-1-phenylethylamine to methyl4-methoxy-cinnamate

A 1 L 3-neck round bottom flask, equipped with a stopper, thermometer,and 250 mL addition funnel with an Ar inlet was charged with(R)-(+)-N-benzyl-1-phenylethylamine hydrochloride (0.132 mol, 32.6 g,1.1 eq based on cinnamate) and the apparatus flushed with Ar 30 min. Thesalt was suspended in dry THF (200 mL) and the mixture cooled to aninternal temperature of −70° C. with a dry ice/acetone bath. To thesuspension was added n-BuLi (2.5 M in hexanes, 0.257 mol, 103 mL, 1.95eq based on amine hydrochloride) from the addition funnel at such a ratethat the internal temperature did not exceed −65° C. The additionrequired 90 min. After completing the addition, the reaction was stirredat −70° C. for 1 hr. A solution of methyl 4-methoxycinnamate (0.120 mol,23 g, 1 eq) in THF (125 mL) was added from the addition funnel over 90min at such a rate that the internal temperature did not exceed −65° C.After completing the addition, the reaction was stirred at −70° C. 2hrs. TLC analysis indicated complete reaction. The reaction was quenchedcold by the addition of 5% citric acid (250 mL) and stirred overnight atRT. In a 2 L separatory funnel, the layers were separated and theorganic washed with 5% citric acid (1×125 mL). The combined aqueous wereextracted with EtOAc (1×200 mL). The combined organics were then washedwith 5% NaHCO₃ (1×150 mL) and brine (1×150 mL) and dried (MgSO₄).Filtration and evaporation to constant weight provided crude product(50.04 g, 103% of theory) as a viscous oil which solidified on standing.Pure material was obtained by triturating and stirring crude productwith heptane (1.5-2 mL/g, 75-100 mL total volume) at RT overnight. Thesolids were collected by suction filtration on a medium frit and thecake washed by flooding with cold heptane (2×50 mL). The solids weredried on the filter to give pure product (28.93 g, 60% yield) as a whitepowder. TLC (4:1 hexane/EtOAc): R_(f)=0.50 (I₂, UV); mp=87-88° C.; ¹HNMR (CDCl₃, 300 MHz): 1.20 (d, 3H, J=6.9 Hz), 2.51 (dd, 1H, J=9.4, 14.8Hz), 2.66 (dd, 1H, J=5.7, 14.8 Hz), 3.45 (s, 3H), 3.67 (ABq, 2H, J=14.7Hz), 3.79 (s, 3H), 3.98 (q, 1H, J=6.8 Hz), 4.37 (dd, 1H, J=5.7, 9.3 Hz),6.86 (d, 2H, J=8.6 Hz), 7.16-7.33 (m, 10H), 7.40 (d, 2H, J=7.3 Hz); MS(FAB): 404

Hydrogenolysis of benzyl groups

The above adduct (0.071 mol, 28 g) was suspended in MeOH (300 mL) andtreated with formic acid (96%, 0.179 mol, 8.25 g, 6.8 mL, 2.5 eq) neatin one portion with stirring. To this suspension was added Degussa typeE101 NE/W 10% Pd/C (50% wet, 0.00179 mol, 3.81 g, 0.025 eq) in oneportion. The resulting mixture was heated at reflux for 1-2 hr untiljudged complete by TLC analysis. The mixture was cooled to RT, thenfiltered on a pad of Celite, washing the flask and pad with MeOH (150mL). The combined filtrates were evaporated to give crude product (15.42g, 102% of theory) as an oil. The crude product was dissolved in i-PrOH(250 mL) and heated to a gentle reflux. D-tartaric acid (0.071 mol,10.76 g, 1 eq) was added as a solid in one portion. Heating wascontinued for 15 min, during which time the salt precipitated as a finewhite solid. The mixture was cooled to RT, then refrigerated overnight.The crystalline salt was collected by suction filtration on a mediumfrit, washing with cold i-PrOH (50-75 mL), and dried on the filter togive product (23 g, 79%). The above salt was converted to the free baseby dissolving in a minimum volume of H₂O (125 mL) and treating thesolution with solid NaHCO₃ until the aqueous was saturated. This wasextracted with EtOAc (3×100 mL). The combined organics were washed withbrine (1×100 mL) and dried (MgSO₄). Filtration and evaporation providedpure product (11.75 g, 78%) as a nearly colorless oil which solidifiedon cooling.

TLC (9:1 CHCl₃/MeOH): R_(f)=0.30 (I₂, UV); HPLC (reverse phase;MeCN/H₂O/TFA gradient): 96% pure, R_(t)=17.9 min; ¹H NMR (CDCl₃, 300MHz): 1.87 (br s, 2H), 2.62 (d, 2H, J=6.9 Hz), 3.64 (s, 3H), 3.76 (s,3H), 4.35 (t, 1H, J=6.9 Hz), 6.84 (d, 2H, J=8.6 Hz), 7.25 (d, 2H, J=8.6Hz); MS (FAB): 210.

¹H NMR: (CDCl₃, 300 MHz, ppm) 7.41-7.28 (m, 5H), 4.18 (q, 2H), 2.65 (d,2H), 2.12 (br, 2H), 1.16 (t, 3H)

¹H NMR (CDCl₃, 300 MHz, ppm) 6.81 (d, 1H, J=1.6 Hz), 6.72 (d, 1H, J=7.9Hz), 6.66 (d, 1H, J=7.9 Hz), 5.85 (s, 2H), 4.22 (1H, dd, J=7.5 Hz and7.3 Hz), 2.47 (2H, dd, J=7.5 Hz and 5.6 Hz), 2.21(s, 2H), 1.35 (9H, s).

¹H NMR (CDCl₃, 300 MHz, ppm) 6.82 (d, 1H, J=1.6 Hz), 6.76 (d, 1H, J=7.9Hz), 6.73 (d, 1H, J=7.9 Hz), 5.89 (s, 2H), 4.29 (1H, dd, J=6.9 Hz and6.8 Hz), 3.63 (3H, s), 2.57 (d, 2H, J=6.9 Hz), 1.75 (s, 2H);

¹H NMR (CDCl₃, 300 MHz, ppm) 6.79-6.78 (m, 3H), 4.32 (t, 1H, J=6.7 Hz),3.75 (s, 3H), 3.72 (s, 3H), 2.52 (d, 2H, J=6.8 Hz), 1.82 (br, 2H), 1.42(s, 9H).;

¹H NMR (CDCl₃, 300 MHz, ppm) 7.20 (d, J=8.6 Hz), 6.80 (d, 2H, J=8.6 Hz),4.30 (t, 1H, 6.8 Hz), 3.71 (s, 3H), 3.60 (s, 3H), 2.57 (d, 2H, J=6.8Hz), 1.91 (s, 2H);

¹H NMR (CDCl₃, 300 MHz, ppm) 7.24 (d, J=8.4 Hz), 6.82 (d, 2H, J=8.4 Hz),4.26 (t, 1H, 6.8 Hz), 3.66 (s, 3H), 2.47 (d, 2H, J=6.6 Hz), 1.41 (s,9H);

¹H NMR (CDCl₃, 300 MHz, ppm) 7.21(dd, 1H, J=8.2 Hz and 8.1 Hz),6.95-6.93 (m, 2H), 6.78 (d, 1H, 6.8 Hz), 4.34 (t, 1H, J=6.7 Hz), 3.79(s, 3H), 2.54 (d, 2H, J=6.9 Hz), 1.74 (s, 2H), 1.40 (s, 9H);

¹H NMR (CDCl₃′ 300 MHz, ppm): 7.34-7.08 (m, 2H), 6.82-6.68 (m, 2H), 4.45(m, 1H), 3.65 (s, 3H), 3.49 (s, 3H), 2.58 (d, 2H), 1.68 (br s, 2H).

¹H NMR (CDCl₃, 300 MHz, ppm) 7.28-7.25 (m, 2H), 7.01 (d, 1H), 4.31(t,1H), 2.50 (d, 2H), 2.01 (br, 2H), 1.41 (s, 9H);

¹H NMR (CDCl₃, 300 MHz, ppm) 6.84 (s,1H), 6.79-6.76 (m, 1H), 4.24-4.19(m, 1H), 4.19 (s, 4H), 2.50 (d, 2H), 1.63 (br, 2H), 1.41 (s, 9H);

¹H NMR (CDCl₃, 300 MHz, ppm) 3.34-3.05 (m, 1H), 2.65-2.58 (m, 2H), 1.65(d, 2H);

¹H NMR (CDCl₃, 300 MHz, ppm) 7.34-7.28 (m, 3H), 7.26-7.15 (m, 3H),3.42-3.15 (m, 1H), 2.71 (dd, 1H, J=5.5 Hz and 13.3 Hz), 2.54 (dd, 1H,J=8.1 Hz and 13.3 Hz), 2.36 (dd, 1H, J=4.2 Hz and 15.7 Hz), 2.20 (dd,J=8.6 and 15.7 Hz), 1.42 (s, 9H).

To Prepare β-13 Amino Acid

1M TMSCl in CH₂Cl₂ (33 ml, 33 mmol) was added to a mixture of(R)-α-methylbenzylamine (3.4 g, 28 mmol) and Et₃N (4 g, 40 mmol) in THF(10 ml) was added and the mixture was allowed to stir for 1 h at roomtemperature. After the solid was removed by filtration, the solution wasconcentrated to afford a liquid. This silylamine (2.4 g, 12.5 mmol) wasdissolved in THF (35 ml) and was cooled to −78° C. To this cooledsolution was added n-BuLi (7.8 ml of 1.6 M solution in hexanes, 12.5mmol) slowly. After stirring for 0.5 h at the temperature, to thereaction mixture was added a solution of t-butyltrans-3-(3-pyridyl)acrylate (2.56 g, 12.4 mmol) in THF (10 ml). Thestirring was continued for another ½ h and the mixture was quenched withsat. NH₄Cl (20 ml) and was allowed to warm up to room temperature andextracted with ether. The combined ether layers were dried (K₂CO₃) andconcentrated to afford an oil. This oil (500 mg) was dissolved inethanol (1.5 ml), t-butanol (15 ml), ammonium formate (1.5 g) and 10%Pd/C (1.2 g) were added. The resulting mixture was heated to reflux for3 h followed by acid and base workup to afford the desired amine β-13(300 mg). FAB-MS=223.

¹HNMR (CDCl₃): δ 7.97(2H, d, J=5.4 Hz), 7.41(2H, d, J=5.4 Hz), 4.40(1H,t, J=4.5 Hz), 3.88(3H,s), 2.55(2H, d, J=4.5 Hz), 1.71(2H, br), 1.39 (9H,s).

General Procedure for Synthesis of M-1, M-2 and M-3

To a solution of the commercially available amino acid (1.5 mmoles) inCH₂Cl₂ (4 ml) and MeOH (1 ml) cooled to 0° C., was added thionylchloride (0.125 ml, 1.65 mmol). The reaction was warmed to 40° C. for 2h, and concentrated to dryness in vacuo to afford the desired aminoester HCl salt.

89% yield; ¹HNMR (DMSO-d⁶′ 300 MHz, ppm): 9.00-8.75 (3H, bm), 7.71 (2H,d, J=7.3 Hz), 7.58 (2H, d, J=7.3 Hz) 4.71 (1 H, bs), 3.64 (3 H,s),3.40-3.06 (2H, m);

85% yield as a tan solid. ¹HNMR (CDCl₃′ 300 MHz, ppm): 7.55-7.05 (6 H,bm), 3.66 (3H, s), 3.65-3.45 (2H, bm), 3.10-2.77 (5H, bm), 2.17-1.95(2H, bm);

84% yield as a pale tan solid; ¹HNMR (CDC₃′ 300 MHz, ppm): 8.1-7.8 (4H,bm), 7.65-7.45 (3H, bm), 5.45 (1H, br), 3.80-3.30 (2H, bm), 3.55 (3H,s).

Procedure C—Synthesis of Coupled Amino Acids

To a solution of ethyl 3-amino-3-phenyl-1-propanoate (or other β-aminoacid ester prepared by Procedure B) (0.50 g, 5.25 mmol) in CH₂Cl₂ (5 ml)was added BocLeuOSu (1.5 g, 4.67 mmol) (CbzLeuOSu is used for the Cbzprotected analog) with cooling and Et₃N (5 drops). The mixture wasstirred at room temperature for 1 h. The reaction mixture was dilutedwith CH₂Cl₂ (10 ml) and washed with 5% citric acid (5 ml×2), 5% NaHCO₃(5 ml) and sat. NaCl (5 ml). The organic layer was dried (Na₂SO₄) andconcentrated to afford 1.26 g (66%) as a white solid.

Procedure D—Synthesis of Deprotected Amino Acids

To a stirred solution of the product of Procedure C (a Boc-Leu-β-aminoacid ester) (41.5 mg, 0.102 mmol) at 0-5° C. in 2 mL of CH₂Cl₂ was added4 mL of TFA. The mixture was allowed to come to room temperature withcontinued stirring for 1 hour. The reaction was concentrated in vacuo,redissolved in CH₂Cl₂, concentrated two more times and placed under highvacuum to remove final traces of TFA. HPLC showed complete conversion totwo new peaks of shorter retention time. The residue can taken up in DMFand TEA added with stirring until basic to litmus in preparation forfurther reaction. A Cbz group is removed using the following method:

The product from Procedure C (where t-butyl3-amino-3-phenyl-1-propanoate and CbzLeuOSu were used) (110 mg, 0.23mmol) in MeOH with a catalytic amount of 10% palladium on charcoal wasstirred overnight under hydrogen at 40 psi. The reaction was filteredthrough Celite® and concentrated in vacuo yielding the free base Leu BOCβ-amino acid (87 mg, quantitative) as a clear oil. ¹H NMR: (CDCl₃, 300MHz, ppm), 7.30 (m, 5H), 5.33 (dd, 1H, J=6, 8.82 Hz), 4.00 (m, 1H) 2.77(dd, 1H J=9, 15 Hz), 2.90 (dd, 1H, J=6, 15 Hz), 1.69 (m, 2H), 1.45 (m,1H), 1.29 (s, 9H), 0.90 (d, 6H, J=6 Hz).

EXAMPLE 1 Synthesis of BIO-1002

A. A stirred solution of cyanoacetic acid (13 mg, 0.15 mmol), EDC (30mg, 0.16 mmol), and HOBt (30 mg, 0.20 mmol) in DMF (0.5 mL) was treatedwith a solution of the amine prepared in Procedure D (52 mg, 0.105 mmol)and diisopropylethylamine (0.30 mL, 1.7 mmol) in DMF (1.0 mL) at roomtemperature. After the solution was stirred for over 18 h, the reactionwas partitioned in ethyl acetate (15 mL) and 60% sat. NaHCO₃ (10 mL).The organic phase was washed with 60% sat. aq. NaHCO₃ (2×10 mL), H₂O (5mL), 5% citric acid (3×10 mL), H₂O (5 mL), and sat. aq. NaCl (10 mL).The organic phase was dried (MgSO₄) and concentrated in vacuo to affordBIO-1002-OEt (27 mg, 69%) as a foam:

¹H NMR (CDCl₃, 300 MHz, ppm) 7.58 (d, 1H), 7.45 (d, 1H), 7.40-7.20 (m,5H), 5.28 (m, 1H), 4.46 (m, 1H), 4.05 (m, 2H), 3.23 (m, 2H), 2.79 (m,2H), 1.78-1.53 (m, 3H), 1.23 (m, 3H), 0.90 (m, 6H).

B. A stirred solution of BIO-1002-OEt (27 mg, 0.072 mmol) in methanol (3mL) was treated with aq. LiOH (1.0 M, 0.25 mL, 0.25 mmol) at roomtemperature for 22 h. The reaction was acidified with trifluoroaceticacid then concentrated in vacuo. The crude products were purified byHPLC to give BIO-1002A (2.5 mg, 10%) and BIO-1002B (4.4 mg, 18%) aswhite solids:

BIO1002A:¹H NMR (CDCl₃, 300 MHz, ppm) 8.08 (d, 1H), 7.87 (d, 1H),7.30-7.16 (m, 5H), 5.25 (m, 1H), 4.37 (m, 1H), 3.36 (s, 2H), 2.75 (m,2H), 1.70-1.45 (m, 3H), 0.90 (m, 6H); HPLC (Gradient A), 16.7 min; MS,m/z 346

BIO1002B: ¹H NMR (CDCl₃, 300 MHz, ppm) 8.00-7.70 (m, 2H), 7.40-7.20 (m,5H), 5.28 (m, 1H), 4.39 (m, 1H), 3.45 (s, 2H), 2.78 (m, 2H), 1.65-1.40(m, 3H), 0.90 (m, 6H); HPLC (Gradient A), 20.6 min; MS, m/z 346.

EXAMPLE 2 Synthesis of BIO-1003

A. The procedure as described Example 1A was performed utilizingcyclohexylacetic acid (22 mg, 0.15 mmol), EDC (30 mg, 0.16 mmol), andHOBt (30 mg, 0.20 mmol), amine from Procedure D (52 mg, 0.105 mmol) anddiisopropylethylamine (0.30 mL, 1.7 mmol) in DMF (1.0 mL) to affordBIO-1003-OEt (32 mg, 71%) as a foam: ¹H NMR (CDCl₃, 300 MHz, ppm)7.42-7.18 (m, 6H), 6.08 (m, 1H), 5.36 (m, 1H), 4.50 (m, 1H), 4.05 (m,2H), 2.81 (m, 2H), 2.11-0.80 (m, 25H).

B. The procedure as described in Example 1B was performed utilizingBIO-1003-OEt (32 mg, 0.074 mmol) and aq. LiOH (1.0 M, 0.25 mL, 0.25mmol) in MeOH (3.0 mL) to give BIO-1003A (3.5 mg, 11%) and BIO-1003B(5.3 mg, 18%) as white solids:

BIO-1003A: ¹H NMR (CDCl₃, 300 MHz, ppm) 7.35-7.16 (m, 5H), 5.23 (m, 1H),4.38 (m, 1H), 2.28 (d, 2H), 2.03 (m, 2H), 1.75-0.80 (m, 22H); HPLC(Gradient A), 34.1 min and 35.3 min (4:1); MS, m/z 403.

BIO-1003B: ¹H NMR (CDCl₃, 300 MHz, ppm) 7.35-7.16 (m, 5H), 5.23 (m, 1H),4.38 (m, 1H), 2.28 (m, 2H), 2.03 (m, 2H), 1.75-0.80 (m, 22H); HPLC(Gradient A), 34.1 min and 35.3 min (1:10); MS, m/z 403.

EXAMPLE 3 Synthesis of BIO-1014

A. Methyl 3-amino-3-phenyl-1-propanoate was coupled with BocLeuOSu bythe method described in Procedure C. This material was subjected to theconditions used in Procedure D1 to give the desired TFA-amine salt.

B. The procedure as described in Example 1A was performed utilizingindole-3-carboxylic acid (19 mg, 0.12 mmol), EDC (26 mg, 0.14 mmol),HOBt (26 mg, 0.17 mmol), amine from Example 3A (44 mg, 0.11 mmol) anddiisopropylethylamine (0.10 mL, 0.56 mmol) in CH₂Cl₂ (5.0 mL) to affordBIO-1014-OMe (25 mg, 52%) as a foam.

C. The same procedure as described in Example 1B was performed utilizingBIO-1014-OMe (25 mg, 0.057 mmol) and aq. LiOH (1.0 M, 0.115 mL, 0.115mmol) in MeOH (5 mL) to give BIO-1014A (5.1 mg, 21%) and BIO-1014B (4.7mg, 20%) as white solids:

BIO-1014A: ¹H NMR (CDCl₃, 300 MHz, ppm) 8.52 (d, 1H), 8.13 (d, 1H), 8.10(d, 1H), 7.81 (d, 1H), 7.46-7.03 (m, 9H), 5.20 (m, 1H), 4.58 (m, 1H),2.69 (m, 2H), 1.75-1.45 (m, 3H), 0.90 (m, 6H); HPLC (Gradient A), 28.1min; MS, m/z 422.

BIO-1014B: ¹H NMR (CDCl₃, 300 MHz, ppm) 8.55 (d, 1H), 8.18 (d, 1H), 8.13(d, 1H), 7.79 (d, 1H), 7.46-7.03 (m, 9H), 5.20 (m, 1H), 4.58 (m, 1H),2.70 (m, 2H), 1.55-1.40 (m, 3H), 0.90 (m, 6H); HPLC (Gradient A), 29.5min; MS, m/z 422.

EXAMPLE 4 Synthesis of BIO-1017

A. The procedure as described in Example 1A was performed utilizing1-phenyl-1-cyclopropanecarboxylic acid (21 mg, 0.13 mmol), EDC (26 mg,0.14 mmol), HOBt (26 mg, 0.17 mmol), amine from Example 3A (44 mg, 0.11mmol) and diisopropylethylamine (0.10 mL, 0.56 mmol) in CH₂Cl₂ (5.0 mL)to afford BIO-1017-OMe (39 mg, 68%) as a foam.

B. The procedure as described in Example 1B was performed utilizingBIO-1017-OMe (39 mg, 0.089 mmol) and aq. LiOH (1.0 M, 0.27 mL, 0.27mmol) in MeOH (2 mL) to give BIO-1017A (10.3 mg, 27%) and BIO-1017B(12.2 mg, 32%) as white solids:

BIO-1017A: ¹H NMR (CD₃SOCD₃, 300 MHz, ppm) 8.46 (d, 1H), 7.40-7.20 (m,10H), 6.30 (d, 1H), 5.09 (m, 1H), 4.33 (m, 1H), 2.62 (m, 2H), 1.50-1.20(m, 5H), 0.98 (m, 2H), 0.82 (m, 6H); HPLC (Gradient A), 33.9 min; MS,m/z 423.

BIO-1017B: ¹H NMR (CD₃SOCD₃, 300 MHz, ppm) 8.55 (d, 1H), 7.48-7.15 (m,10H), 6.30 (d, 1H), 5.08 (m, 1H), 4.35 (m, 1H), 2.63 (m, 2H), 1.48-1.15(m, 5H), 1.10-0.88 (m, 2H), 0.85-0.64 (m, 6H); HPLC (Gradient A), 33.9min and 34.5 min (1:9); MS, m/z 423.

EXAMPLE 5 Synthesis of BIO-1022

A. The procedure as described in Example 1A was performed utilizing2-naphthylacetic acid (20 mg, 0.11 mmol), EDC (25 mg, 0.13 mmol), HOBt(25 mg, 0.16 mmol), amine from Example 3A (42 mg, 0.10 mmol) anddiisopropylethylamine (0.10 mL, 0.56 mmol) in DMF (2.0 mL) to affordBIO-1022-OMe (36 mg, 70%) as a foam.

B. The procedure as described in Example 1B was performed utilizingBIO-1022-OMe (36 mg, 0.078 mmol) and aq. LiOH (1.0 M, 0.50 mL, 0.50mmol) in MeOH (3 mL) to give BIO-1022A (1.7 mg, 4.8%) and BIO-1022B (6.8mg, 19%) as white solids:

BIO-1022A: ¹H NMR (CDCl₃, 300 MHz, ppm) 7.90-7.17 (m, 12H), 5.30 (t,1H), 4.45 (m, 1H), 2.79 (m, 2H), 1.68-1.33 (m, 3H), 0.87 (d, 6H); HPLC(Gradient A), 25.7 min; MS, m/z 447.

BIO-1022B: ¹H NMR (CDCl₃, 300 MHz, ppm) 7.90-7.17 (m, 12H), 5.35 (t,1H), 4.49 (m, 1H), 2.79 (d, 2H), 1.58-1.33 (m, 3H), 0.82 (m, 6H); HPLC(Gradient A), 25.7 min and 26.4 min (1:9); MS, m/z 447.

EXAMPLE 6 Synthesis of BIO-1029

A. t-Butyl 3-amino-3-phenyl-1-propanoate was coupled with BocLeuOSuusing the method described in Procedure C. This material was subjectedto the conditions of Procedure D2 to give the desired amine salt.

B. The procedure as described in Example 1A was performed utilizing4-(2-aminobenzamido)-phenylacetic acid (18 mg, 0.067 mmol), EDC (13 mg,0.067 mmol), and HOBt (13 mg, 0.085 mmol), amine from Example 6A (18 mg,0.054 mmol) and diisopropylethylamine (0.048 mL, 0.27 mmol) in DMF (0.5mL) to afford NH₂-BIO-1029-OtBu (32 mg, 100%) as an oil:

¹H NMR (CDCl₃, 300 MHz, ppm) 7.65-7.43 (m, 4H), 7.40-7.10 (m, 9H), 6.72(m, 2H), 6.49 (d, 1H), 5.28 (m, 1H), 4.45 (m,1H), 3.52 (s, 2H), 2.68 (m,2H), 2.00 (bs, 2H),1.65-1.15 (m, 13H), 0.85 (m, 6H).

C. A solution of NH₂-BIO-1029-OtBu (16 mg, 0.027 mmol) intrifluoroacetic acid (1 mL) was stirred at room temperature for 45 minand then concentrated. The crude product was purified by HPLC to affordNH₂-BIO-1029 (3.4 mg, 26%) as a white solid: MS, m/z 531.

D. A solution of NH₂-BIO-1029 (3.4 mg, 0.0064 mmol), methyl isocyanate(3 drops), and diisopropylethylamine (1 drop) in CH₂Cl₂ (0.30 mL) wasstirred at room temperature for 18 h and then concentrated in vacuo. Thecrude product was purified by HPLC to afford BIO-1029 (2.6 mg, 69%) as awhite solid: ¹H NMR (CD₃SOCD₃, 300 MHz, ppm) consistent with structure;HPLC (Gradient A), 28.2 min; MS, m/z 588.

EXAMPLE 7 Synthesis of BIO-1032

A. The procedure as described in Example 1A was performed utilizing3-amino-phenylacetic acid (29 mg, 0.19 mmol), EDC (44 mg, 0.23 mmol),and HOBt (44 mg, 0.29 mmol), amine from Example 6A (49 mg, 0.15 mmol)and diisopropylethylamine (0.17 mL, 0.95 mmol) in DMF (1.0 mL) to affordNH₂-BIO-1032-OtBu (22 mg, 31%) as a foam after flash chromatography(SiO₂, 60% ethyl acetate-hexane) ¹H NMR (CDCl₃, 300 MHz, ppm) 7.45-7.05(m, 7H), 6.75-6.50 (m, 3H), 5.97 (d, 1H), 5.30 (m, 1H), 4.46 (m, 1H),3.50 (s, 2H), 2.71 (m, 2H), 1.70-1.39 (m, 3H), 1.33 (s, 9H), 0.84 (m,6H).

B. A mixture of NH₂-BIO-1032-OtBu (7.0 mg, 0.015 mmol), phenylsulfonylchloride (1.7 μL, 0.014 mmol), and diisopropylethylamine (5.4 μL, 0.030mmol) in CH₂Cl₂ was stirred at room temperature for 18 h. The reactionmixture was concentrated in vacuo and the residue diluted with ethylacetate. The organic solution was washed with 60% sat. aq. NaHCO₃ (2×),H₂O, 5% citric acid (3×), H₂O , and sat. aq. NaCl, dried (MgSO₄) andconcentrated. The residue (9 mg) was stirred in trifluoroacetic acid (1mL) at room temperature for 30 min before concentrating in vacuo. Theresulting crude product was purified by HPLC to afford BIO-1032 (3.9 mg,47%) as a white solid: ¹H NMR (CD₃SOCD₃, 300 MHz, ppm) 8.52 (d, 1H),8.17 (d, 1H), 7.75 (d, 2H), 7.61-7.45 (m, 3H), 7.35-6.85 (m, 9H), 5.13(m, 1H), 4.28 (m, 1H), 3.40 (m, 2H), 2.65 (bs, 2H), 1.50-1.12 (m, 3H),0.79 (d, 3H), 0.71 (d, 3H) HPLC (Gradient B), 18.7 min; MS, m/z 552.

EXAMPLE 8 Synthesis of BIO-1093

A. To a stirred solution of the Boc-protected amine product of ProcedureC (41.5 mg, 0.102 mmol) at 0-5° C. in 2 mL of CH₂Cl₂ was added 4 mL ofTFA. The mixture was allowed to come to room temperature with continuedstirring for 1 hour. The reaction was concentrated in vacuo, redissolvedin CH₂Cl₂, concentrated two more times and placed under high vacuum toremove final traces of TFA. HPLC showed complete conversion to two newpeaks of shorter retention time.

B. The material from Example 8A was redissolved in 0.75 mL DMF, cooledto 0-5° C. and DIEA was added until the mixture was basic to litmus andthe ice bath was removed. This material combined with 4-nitrophenylacetic acid (16.5 mg, 0.091 mmol), HOBt (20.4 mg, 0.151 mmol) and EDC(19.4 mg, 0.101 mmol) under conditions described in Example 1A to yieldBIO 1093-OEt (21.4 mg, 50%) as a clear oil.

C. A solution of BIO 1093-OEt (21.4 mg, 0.053 mmol) in 1 ml of MeOH wasstirred overnight at room temperature with 1N LiOH (130 μl, 0.13 mmol).The mixture was acidified (red to litmus) with TFA and concentrated invacuo. Pure isomers were resolved via preparative HPLC followed bylyophilization. Repeated dissolution in 50/50 MeOH/CH₂Cl₂ and in vacuoconcentration followed by 24 hours under high vacuum provided BIO-1093(3 mg, 13%) of each isomer as white amorphous solids: Isomer A: ¹H NMR:(CDCl₃, 300 MHz, ppm), 8.09 (d 2H J=8.2 Hz), 7.38 (d, 2H, J=8.21 Hz),7.15 (s, 5H), 5.21 (m, 1H), 4.32 (m, 1H), 3.28 (s, 1H), 2.67 (m, 2H),1.40 (M, 3H), 0.75 (dd, 6H J=6.9, 7.6 Hz). FAB: 442 (M+H)⁺, 464 (M+Na)⁺Mw 441.43. HPLC: Gradient 1 single peak >99% 19.5 min. Tlc: 10%MeOH/CH₂Cl₂ Rf=0.25, EtOAc plus 1% HOAc R_(f)=0.35.

Isomer B: ¹H NMR: (CDCl_(3, 300) MHz, ppm), 8.0 (d, 2H, J=9.7 Hz), 7.56(d, 1H J=8.0 Hz), 7.73 d, 2H J=9.7 Hz), 7.07 (s, 5H), 5.15 (t, 1H, J=5.5Hz), 4.29 (m, 1H), 3.45 (s, 2H), 2.65 (m, 2H), 1.45 (m, 3H), 0.78 (dd,6H, J=6.9, 4.8 Hz). FAB: 442 (M+H)⁺, 464(M+Na)⁺, MW 441.43. HPLC: Singlepeak >99%, 19.3 min. Tlc: 10% MeOH/CH₂Cl₂ R_(f)=0.29, EtOAc plus 1% HOAcR_(f)=0.55.

EXAMPLE 9 Synthesis of BIO-1099

A. The amine from Example 3A (50.0 mg, 0.127 mmol) was subjected to theconditions described in Example 8B using diphenylacetic acid (25.6 mg,0.121 mmol), HOBt (26 mg, 0.19 mmol), and EDC (27 mg, 0.14 mmol) in DMFto afford BIO 1099-OMe (49.2 mg, 83%) as a clear viscous oil.

B. BIO-1099-OMe (49 mg, 0.1 mmol) was saponified and purified asdescribed in Example 8C to provide BIO-1099A (7 mg, 15%) and BIO-1099B(5 mg, 11%) as white amorphous solids. Isomer A: ¹H NMR: (CDCl₃, 300MHz, ppm), 7.95 (d, 1H 8 Hz), 7.19 (m, 15H), 6.95 (d, 1H 8 Hz), 5.25 (t,1H, J=3.2), 4.84 (s, 1H), 4.41 (m, 1H), 2.70 (dd, 2H, J=2.5, 1.3 Hz),1.41 (m, 3H), 0.79 (dd, 6H, (J=6 Hz). FAB: (M+H)⁺ 474, (M+Na)⁺ 496 MW472.54 HPLC: 1 peak; 100% pure; 30.074 min. Tlc: 10% MeOH/CH₂Cl₂R_(f)=0.33; 50/50 EtOAc/Hex, 1% HOAc R_(f)=0.45

Isomer B ¹H NMR: (CDCl₃, 300 MHz, ppm) 7.72 (d, 1H, 8 Hz), 7.22 (m,15H), 5.31 (t, 1H, 1.2 Hz), 6.70 (d, 1H 8 Hz), 4.93 (s, 1H), 4.60 (m,1H), 2.68 (s, 1H), 2.65 (m, 2H ), 1.35 (m, 3H), 0.61 (dd, 6H, J=2.5, 1.3Hz). FAB: 473 (M+H)⁺, 495 (M+Na)⁺; MW 472.54 HPLC: 1 Peak; 100%; 30.38min. Tlc: 10% MeOH/CH₂Cl₂ R_(f)=0.33, 50/50 EtOAc/Hex plus 1% HOAcR_(f)=0.38.

EXAMPLE 1 Synthesis of BIO-1100

A. The amine salt described in Example 6A (prepared from 40.5 mg, 0.093mmol of Boc protected material) was taken up in 1.0 mL of DMF and TEAwas added with stirring until basic to litmus.

B. The method described in Example 1A was performed using2-bromo-5-methoxy-4-hydroxy phenyl acetic acid (23.1 mg, 0.089 mmol),HOBt (18.9 mg, 0.14 mmol), EDC (19.6 mg, 0.10 mmol) in 1.0 ml DMF andfree amine prepared in Example 10A to give a white solid (49 mg,quantitative). An aliquot was purified by preparative reverse phase HPLC(gradient 2), lyophilized and dried by repeatedly dissolving in 50/50MeOH/CH₂Cl₂ and concentrated under reduced pressure to yield BIO-1100(1.8 mg) as an amorphous white solid. ¹H NMR: (CDCl₃ 300 MHz, ppm), 7.25(s, 5H), 7.05 (s, 1H), 6.30 (s, 1H), 5.28 (m, 1H), 3.81 (s, 3H), 3.59(s, 2H), 2.77 (m, 2H), 1.45 (m, 3H), 0.82 (dd, 6H J=2.5, 1.2). FAB:(M+H)⁺ 521, 523; (M+Na)⁺ 543, 545; MW 521.44 HPLC: Major peak at 29.1min; >97% purity. Tlc: 10% MeOH/CH₂Cl₂ R_(f)=0.16; 50/50 EtOAc/Hex plus1% HOAc R_(f)=0.28

EXAMPLE 11 Synthesis of BIO-1106

A. To a solution of 6-aminohexanoic acid (1.0 g, 7.6 mmol) in dioxane (6ml) and water (6 ml) containing TEA (1.7 ml, 11.25 mmol) was addedBOC-ON (2.1 g, 8.4 mmol, Aldrich). After stirring for 3 h at roomtemperature, the reaction was diluted with water (20 ml) and washedtwice with ethyl acetate (10 ml). The aqueous was then acidified toPH=1-2 with 1N HCl and the aqueous layer extracted five times with ethylacetate, dried over Na₂SO₄ and concentrated to afford 1106-1 (842 mg,51%). ¹HNMR (CDCl₃′ 300 MHz, ppm): 4.61 (1H, bs), 3.15-2.95 (4H, bm),2.55-2.23 (4 H, m), 1.65-1.50 (4 H), 1.46 (9 H), 1.45-1.30 (2 H, m).

B. t-Butyl 3-amino-3-phenyl-1-propanoate was coupled with CbzLeuoSu asdescribed in Procedure C. This material was subjected to the conditionsof Procedure D2 to give the desired free amine.

C. N-Boc 6-aminohexanoic acid (prepared in Example 11A) (17.3 mg, 0.075mmol), HOBt (15.2 mg, 0.11 mmol) and EDC (17.3 mg, 0.09 mmol) werestirred in 0.5 ml of DMF at room temperature for 1.5 hours. The freeamine from Example 11B (25 mg, 0.075 mmol) in 0.5 ml of DMF was added tothe stirred solution of activated ester along with two drops of TEA sothat the reaction was basic to litmus. After several hours the reactionwas determined to be incomplete by HPLC. Small portions ofN-Boc-6-aminohexanoic acid, HOBt, and EDC were then added to drive thereaction to completion. Purification, as detailed in Example 8C,provided BIO 1106 Boc t-butyl ester (26 mg, 63%) as a clear viscous oil.¹H NMR: (CDCl₃ 300 MHz, ppm), 7.40 (d, 1H, 8 Hz), 7.32-7.25 (m, 5H),6.30 (d, 1H, J=8 Hz), 5.30 (q, 1H, J=7 Hz), 4.49 (m, 1H), 3.09 (bs, 2H),2.79 (dd, 1H, J=8, 15 Hz), 2.69 (dd, 1H, J=7, 15 Hz), 2.20 (t, 2H J=8Hz), 1.69-1.39 (m, 9H), 1.42 (s, 9H), 1.29 (s, 9H), 0.88 (m, 6H). HPLC:1 peak, 100% purity at 28.3 min.

Both t-butyl protecting groups of BIO 1106 Boc t-butyl ester wereremoved as described in Example 10A. The resulting residue was stirredin 0.5 ml of DMF, made basic to litmus by the addition of two drops ofTEA, followed by phenyl isocyanate (13.6 mg, 0.3 mmol) and stirredovernight. The reaction mixture was purified as detailed in Example 10Bresulting in BIO-1106 (3.5 mg, 29%) as a beige amorphous solid. ¹H NMR:(CDCl₃, 300 MHz, ppm), 7.97 (d, 1H, 8 Hz), 7.22 (m, 11H), 6.91 (t, 1HJ=8 Hz), 5.30 (m, 1H), 4.33 (m, 1H), 3.12 (m, 6H), 2.63 (m, 2H), 2.13(t, 2H, J=6 Hz), 1.41 (bm, 9H), 0.80 (m, 6H). FAB: (M+H)⁺ 511, (M+Na)⁺533; MW 510.59. HPLC: 1 peak; 100% at 19.4 min. Tlc: 15% MeOH/CH₂Cl₂R_(f)=0.32, 10% MeOH/EtOAc plus 1% HOAc R_(f)=0.31.

EXAMPLE 12 Synthesis of BIO-1142

(±)-1-Benzocyclobutene carboxylic acid (16.3 mg, 0.11 mmol), HOBt (22.4mg, 0.165 mmol), and EDC (23.7 mg, 0.121 mmol) were stirred in 0.5 mlDMF at room temperature for 45 minutes to give the activated ester. Theproduct of Example 10A (15.3 mg, 0.055 mmol) was added to the activatedester and the mixture stirred for two hours. Filtration and preparativeHPLC purification, as described in Example 10B, yielded BIO-1142 isomerA (4.4 mg, 70%) and BIO-1142 isomer B (4.9 mg, 22%) as white amorphoussolids. BIO-1142 isomer A: ¹H NMR: (CDCl₃, 300 MHz,ppm), 7.79 (d, 1H J=8Hz), 7.31-7.05 (m, 9H), 6.81 (d, 1H J=8 Hz), 5.24 (m, 1H), 4.36 (m, 1H),4.15 (m, 1H), 3.00-3.50 (bm, 11H), 2.70 (m, 2H), 1.43 (m, 3H), 0.70 (m,6H). FAB: (M+H)⁺ 409 (M+Na)⁺ 431; MW 408.46. HPLC: Major peak at 20.2min; >99% purity. Tlc: 10% MeOH/CH₂Cl₂ R_(f)=0.46, EtOAc plus 1% HOAcR_(f)=0.53.

BIO-1142 isomer B: ¹HNMR: (CDCl₃, 300 MHz, ppm), 7.92 (d, 1H, J=8 Hz),7.31-7.05 (m, 9H), 6.91 (d, 1H, J=8z), 5.25 (m, 1H), 4.38 (m, 1H), 4.14(m, 1H), 3.28 (m, 2H), 2.72 (m, 2H), 1.42 (m, 3H), 0.77 (m, 6H). FAB:(M+H)⁺ 409 (M+Na)⁺ 431; MW 408.46. HPLC: Major peak at 20.62 min; >96%purity. Tlc :10% MeOH/CH₂Cl₂ R_(f)=0.52; EtOAc plus 1% HOAc R_(f)=0.54.

EXAMPLE 13 Synthesis of BIO-1189

(±)-1-indancarboxylic acid (6.2 mg, 0.038 mmol), HOBt (7.7 mg, 0.057mmol), and EDC (8.0 mg, 0.042 mmol) were stirred in 0.5 ml DMF at roomtemperature for two hours. The free amine prepared in Example 11B wastreated with TFA and this material (10 mg, 0.038 mmol) was then addedand the mixture stirred overnight. Filtration and preparative HPLCpurification as described in Example 10B yielded BIO-1189 isomer A (lessthan 1 mg) and isomer B (2 mg, 12%) as white amorphous solids.

BIO-1189 isomer A: ¹H NMR: (CDCl₃, 300 MHz, ppm), 7.3-7.1 (m, 12H), 5.32(m, 1H), 4.48 (m, 1H), 3.91 (t, 1H J=6.6 Hz), 3.1-2.7 (m, 3H), 2.5-2.2(m, 1H), 1.6-1.4 (m, 3H), 0.85 (m, 6H)

FAB: (M+H)⁺, 423 (M+Na)⁺ 445; MW 422.5.

HPLC: Major peak 21.2 min.; >97% purity.

Tlc: 5% MeOH/CH₂Cl₂ R_(f)=0.19; EtOAc plus 1% HOAc R_(f)=0.73.

BIO-1189 isomer B: ¹H NMR: (CDCl₃, 300 MHz, ppm), 7.7(d, 1H, J=8 Hz),7.45-7.1 (m, 9H), 6.65 (d, 1H, J=8 Hz), 5.33 (m, 1H), 4.48 (m, 1H), 3.90(t, 1H, J=6.6 Hz), 3.1-2.8 (m, 3H), 2.45-2.3 (m, 2H), 1.48 (m, 3H), 0.80(m, 6H).

FAB: (M+H)⁺ 423 (M+Na)⁺ 445; MW 422.5 .

HPLC: Major peak 21.5 min; >94% purity.

Tlc: 5% MeOH/CH₂Cl₂ R_(f)=0.12, EtOAc plus 1% HOAc R_(f)=0.60.

EXAMPLE 14 Synthesis of BIO-1006

A. Amine β-3 was coupled with BocLeuOSu according to Procedure C(product recrystallized from diethyl ether) and deprotected according toProcedure D to give the desired TFA-amine salt.

¹H-NMR (300 MHz, CDCl₃) for BOC amine: 0.90 (m, 6H), 1.42 (9H),1.55-1.75 (m, 3H), 2.8 (m, 2H), 3.61 (s, 3H), 4.05 (m, 1H), 4.83 (m,1H), 5.26 (m, 1H), 5.92 (s, 2H), 6.68-6.78 (m, 3H), 7.06 (d, 1H).

1H-NMR (300 MHz, CDCl₃) for TFA-amine: 0.83 (d, 3H), 0.87 (d, 3H), 1.50(m, 1H), 1.63 (bt, 2H), 2.73-2.92 (m, 2H), 3.63 (s, 3H), 4.27 (bs, 1H),5.26 (m, 1H), 5.95 (s, 2H), 6.66-6.78 (m, 3H), 7.58 (bs, 3H), 8.02 (d,1H).

B. A solution of amine-TFA salt of Example 14A (24 mg) in CH₂Cl₂ wasadded to 4-hydroxyphenylacetic acid succinimidyl ester (14 mg, 1.1 eq)and stirred at room temperature for about 2 hours. The reaction mixturewas washed with 5% citric acid (2×), sat. aq. NaHCO₃ (2×) and brine(1×), dried (Na₂SO₄), filtered and concentrated to give 28 mg of crudeBIO-1006 methyl ester. ¹H-NMR: (300 MHz, CDCl3) 0.82 (6 H), 1.35-1.58 (3H), 2.62-2.82 (2 H), 3.48 (2 H), 3.57 (3 H), 4.41 (1 H), 5.70 (1 H),5.89 (2 H), 6.08 (1 H), 6.65-6.75 (5 H), 7.04 (2 H), 7.22 (1 H).

C. Crude BIO-1006 methyl ester in MeOH was added to 1 N LiOH and stirredat room temperature for about 1 hour. The reaction mixture wasneutralized by trifluoroacetic acid and purified by HPLC. The cleanfraction was collected and dried to give BIO-1006.

¹H-NMR (300 MHz, CDCl₃): 0.73 (d, J=6 Hz, 3H), 0.80 (d, J=6 Hz, 3H),1.35 (bt, 2 H), 1.45 (m, 1 H), 2.40 (m, 2H), 3.22-3.38 (m, 2H), 4.23(bq, 1H), 5.02 (m, 1H), 5.93 (s, 2H), 6.65 (d, J=8 Hz, 2H), 6.68-6.80(m, 2H), 6.83 (s, 1H), 7.03 (d, J=Hz, 2H), 8.11 (bd, 1H).

Mass Spec. M/z=457

EXAMPLE 15 Synthesis of BIO-1050

A. To a suspension of 4-amino phenylacetic acid (9 g, 60 mmol) andN-(benzyloxycarbonyloxy)-succinimide (15 g, 60 mmol) in CH₂Cl₂ was addedenough triethylamine to form a homogeneous solution. The mixture wasstirred at room temperature for 30 min and then CH₂Cl₂ was removed byrotavapor. The resulting residue was dissolved in water and acidifiedwith 5% HCl. The solid thus formed was filtered and washed with 5% HCl,water, and diethyl ether to give 12 g (70%) of Cbz-aminophenylaceticacid as a brownish powder. ¹H-NMR (300 MHz, DMSO-d6): 3.48 (s, 2H), 5.13(s, 2H), 7.14 (d, 2H), 7.29-7.45 (m, 7H), 9.73 (s, 1H).

B. The method of Example 1A was performed using Cbz-aminophenylaceticacid from Example 15A (342 mg, 1.2 mmol) in DMF, HOBT (275 mg, 1.8mmol), EDC (276 mg, 1.44 mmol), and a solution of free amine prepared inExample 14A (432 mg, 0.94 mmol) in DMF to give the coupled product whichwas used without further purification.

C. The product of Example 15B was subjected to hydrogenation (H₂, 50psi, 10% Pd/C, MeOH/H₂O, overnight). The reaction mixture was filteredthrough a pad of Celite®, and concentrated to give 0.4 g (90%) of freeamine as a brown powder. ¹H-NMR (300 MHz, CDCl₃) for (F): 0.82 (m, 6H),1.30-1.62 (m, 3H), 2.62-2.82 (m, 2H), 3.45 (s, 2H), 3.57 (s, 3H), 4.37(m, 1H), 5.18 (m, 1H), 5.91 (s, 2H), 6.65-6.80 (m, 5H), 7.02 (d, 2 H).

D. To a solution of free amine from Example 15C (22 mg) in CH₂Cl₂ wasadded phenylisocyanate (8 mg, 1.5 eq) with one drop of triethylamine.The solution was then stirred at room temperature for 2 hours. Afterdiluting with ethyl acetate (15 mL), the mixture was washed with 5%citric acid (2×), sat. aq. NaHCO₃ (2×) and brine (1×), dried (Na₂SO₄),filtered and concentrated to give the crude phenylureamethyl ester.

E. The crude phenylureamethyl ester was dissolved in MeOH and 1 N LiOHwas added at 0° C. and mixture was stirred at room temperature for 2 h.After neutralization with trifluoroacetic acid, the reaction mixture waspurified by HPLC. The pure fraction was collected and dried to giveBIO-1050. ¹H-NMR: (300 MHz, DMSO-D6): 0.76 (d, 3H), 0.80 (d, 3H),1.30-1.50 (m, 3H), 2.52-2.72 (m, 2H), 3.28-3.50 comp, 2H), 4.30 (m, 1H),5.06 (m, 1H), 5.97 (s, 2H), 6.70 (d, 1H), 6.79-6,87 (m, 2H), 6.95 (t,1H), 7.13 (d, 2H), 7.25 (t, 2H), 7.85 (d, 2H), 7.43 (d, 2H), 8.12 (d,1H), 8.40 (d, 1H), 8.60 (s, 1H), 8.66 (s, 1H). Mass Spec: M/z=575.

EXAMPLE 16 Synthesis of BIO-1068

The procedure of Example 15D was followed utilizing cyclohexylisocyanatefor phenylisocyanate. The resulting product was hydrolyzed as describedin Example 15E and the pure fraction from HPLC purification wascollected and dried to give BIO-1068.

¹H-NMR (300 MHz, DMSO-d6): 0.73 (d, J=6 Hz, 3H), 0.80 (d, J=6 Hz, 3H),1.05-1.85 (m, 13H), 2.50-2.75 (m, 2H), 3.23-3.50 (m, 3H), 4.28 (bq, 1H),5.05 (bq, 1H), 5.95 (bs, 2H), 6.02 (d, J=8 Hz, 1H), 6.72 (bd, 1H), 6.71(d, J=8 Hz, 1H), 6.84 (bs, 1H), 7.08 (d, J=8 Hz, 2H), 7.25 (d, J=8 Hz,2H), 8.07 (d, J=8 Hz, 1H), 8.20 (s, 1H), 8.40 (d, J=8 Hz, 1H).

Mass Spec. M/z=581.

EXAMPLE 17 Synthesis of BIO-1079

The procedure of Example 15D was followed utilizing2-methoxyphenylisocyanate for phenylisocyanate. The resulting productwas hydrolyzed as described in Example 15E and the pure fraction fromHPLC purification was collected and dried to give Bio-1079. ¹H-NMR (300MHz, DMSO-d6): 0.75 (d, 3H), 0.80 (d, 3H), 1.30-1.50 (m, 3H), 2.50-2.72(m, 2H), 3.30-3.45 (m, 2H), 3.85 (s, 3H), 4.28 (m, 1H), 5.06 (m, 1H),5.96 (bs, 2H), 6.69-7.02 (m, 8H), 7.13 (d, 2H), 7.34 (d, 2H), 8.05-8.15(m, 3H), 8.42 (bd, 1H), 8.87 (s, 1H), 9.13 (s, 1H). Mass Spec. M/z=605.

EXAMPLE 18 Synthesis of BIO-1082

A. Triethylamine was added to a solution of the TFA-amine salt preparedin Procedure D (43 mg) in CH₂Cl₂ at 0° C. until pH reached 9.0 wasreached, followed by the addition of 4-phenylbutyryl chloride (26 mg).After stirring at room temperature for 2 h, the reaction mixture wasdiluted with ethyl acetate (20 mL) and then washed with 5% citric acid(2×), sat. aq. NaHCO₃ (2×) and brine (1×), dried (Na₂SO₄), filtered andconcentrated to give the desired product as an ethyl ester.

B. The crude ethyl ester was dissolved in MeOH, 1 N LiOH was added at 0°C. and the mixture was stirred at room temperature for 2 h. Afterneutralization with trifluoroacetic acid, the reaction mixture waspurified by HPLC. Two diastereomers were separated and the purefractions were collected and dried to give Bio-1082-A and Bio-1082-B.

Bio-1082-B ¹H-NMR (300 MHz, DMSO-d6): 0.79 (d, 3H), 0.83 (d, 3H),1.29-1.37 (m, 2H), 1.47 (m, 1H), 1.70-1.83 (m, 2H), 2.08-2.17 (m, 2H),2.48-2.58 (m, 2H), 2.67 (bt, 2H), 4.31 (m, 1H), 5.03 (m, 1H), 7.12-7.32(m, 10H), 7.90 (d, 1H), 8.45 (d, 1H). Mass. Spec. M/z=425.

EXAMPLE 19 Synthesis of BIO-1148

A. Amine β-13 was coupled with BocLeuOSu using the method described inprocedure C. This material was subjected to the conditions of ProcedureD1 to give the desired amine salt 1148-1.

B. To a solution of 4-hydroxyphenylacetic acid (3.0 g, 20 mmol) in DMFwas added HOBT (3.7 g, 24 mmol) followed by EDC (4.2 g, 22 mmol) and themixture was stirred at room temperature for 30 min. N-hydroxysuccinimide(2.3 g, 20 mmol) was added and stirred at room temperature overnight.The resulting mixture was diluted with ethyl acetate (150 ml), extractedwith 5% citric acid (2×), saturated NaHCO₃ (2×) and brine (1×) and wasdried over anhydrous Na₂SO₄. Following removal of the solvent in vacuothe product was dissolved in CH₂Cl₂ and precipitated with hexanes toafford 4-hydroxyphenylacetic acid succinimidyl ester (3.9 g, 78%). ¹HNMR (300 MHz, DMSO-d6): 2.79 (s, 4 H), 3.93 (s, 2 H), 6.72 (d, J=8.5 Hz,2 H), 7.12 (d, J=8.5 Hz, 2 H), 9.41 (s, 1 H).

C. Amine salt 1148-1 was hydrolysed under MeOH/aqueous LiOH conditionsto give an acid. A solution of this acid, triethylamine, and4-hydroxyphenylacetic acid-OSu (prepared in Example 19B) in CH₂Cl₂ wasstirred at room temperature for 1 h. The reaction mixture was purifiedby HPLC and the pure fraction was collected and dried to give Bio-1148as a mixture of two diastereomers. ¹H-NMR (300 MHz, DMSO-d6): 0.70-0.90(m, 6H), 1.29-1.63 (m, 3H), 2.73-2.85 (m, 2H), 3.17-3.40 (m, 2H),4.15-4.30 (m, 1H), 5.12-5.28 (m, 1H), 6.58-6.68 (m, 2H), 6.94-7.06 (m,2H), 7.54-7.67 (m, 1H), 7.93-8.16 (m, 2H), 8.53-8.75 (m, 3 H).

Mass. Spec. M/z=414.

EXAMPLE 20 Synthesis of BIO-1168

The procedure that was used in Example 15D was followed utilizing3-methylphenylisocyanate for phenylisocyanate. The resulting product washydrolyzed as described in Example 15E and the pure fraction from HPLCpurification was collected and dried to give Bio-1168. ¹H-NMR (300 MHz,DMSO-d6): 0.76 (d, 3H), 0.82 (d, 3H), 1.30-1.52 (m, 3H), 2.28 (s, 3H),2.54-2.70 (m, 2H), 3.35-3.48 (m, 2H), 4.28 (m, 1H), 5.07 (m, 1H), 5.96(m, 2H), 6.68-6,86 (m, 4H), 7.10-7.25 (m, 4H), 7.30 (s, 1H), 7.35 (d,2H), 8.11 (d, 1H), 8.44 (d, 1H), 8.63 (s, 1H), 8.67 (s, 1H). Mass Spec.M/z=589.

EXAMPLE 21 Synthesis of BIO-1179

The procedure that was used in Example 15D was followed utilizing2-methylphenylisocyanate for phenylisocyanate. The resulting product washydrolyzed as described in Example 15E and the pure fraction from HPLCpurification was collected and dried to give Bio-1179. ¹H-NMR (300 MHz,DMSO-d6): 0.75 (d, 3H), 0.80 (d, 3H), 1.27-1.51 (m, 3H), 2.23 (s, 3H),2.62 (m, 2H), 3.40 (m, 2H), 4.28 (m, 1H), 5.06 (m, 1H), 5.98 (bs, 2H),6.71 (bd, 1H), 6.80 (d, 1H), 6.83 (bs, 1H), 6.92 (bt, 1H), 7.05-7.20 (m,4H), 7.38 (d, 2H), 7.82 (d, 1H), 7.87 (s, 1H), 8.10 (d, 1H), 8.42 (d,1H), 8.93 (s, 1H). Mass Spec. M/z=589.

EXAMPLE 22 Synthesis of BIO-1195

A. Amine β-9 was coupled with BocLeuOSu according to Procedure C to givethe desired product.

¹H-NMR (300 MHz, CDCl₃): 0.90 (m, 6H), 1.32 (s, 9H), 1.42 (s, 9H),1.58-1.90 (m, 3H), 2.61-2.80 (m, 2H), 4.08 (m, 1H), 4.89 (bd, 1H), 5.37(bq, 1H), 6.95-7.15 (m, 3H), 7.45 (bd, 1H).

B. The product of Example 22A was treated with TFA as described inProcedure D to give the corresponding TFA-amine salt 1195-2.

C. A mixture of 4-amino-phenylacetic acid (10.0 g, 66.1 mmol) and 98%phenyl isocyanate (8.27 g, 68.0 mmol) in ethyl acetate (100 mL) wasstirred at RT for 1 h then refluxed for 1.5 h. The mixture was allowedto cool to RT and the product was filtered, washed with ethyl acetate,methanol, and then ether affording phenylureaphenylacetic acid 1195-3(17.5 g, 98%) as a white powder. ¹HNMR (DMSO-d⁶, 300 MHz, ppm):8.72-8.64 (m, 2H), 7.44 (d, 2H), 7.36 (d, 2H), 7.28 (d, 2H), 7.16 (d,2H), 6.96 (t, 1H), 3.52 (s, 2H). FAB-MS=272.

D. A solution of phenylureaphenylacetic acid 1195-3, HOBT, and EDC inDMF was stirred at room temperature for 30 min and then the free amineprepared from the product of Example 22B and TEA treatment was added.After stirring at room temperature overnight, the reaction mixture waspurified by HPLC and the pure fraction was collected and dried to giveBio-1195.

¹H-NMR (300 MHz, DMSO-d6): 0.71 (d, 3H), 0.78 (d, 3H), 1.25-1.46 (m,3H), 2.56-2.72 (m, 2H), 3.26-3.41 (m, 2H), 4.21 (bq, 1H), 5.07 (bq, 1H),6.90 (bt, 1H), 7.02-7.14 (m, 3H), 7.17-7.42 (m, 8H), 8.10 (d, 1H), 8.47(d, 1H), 8.58 (s, 1H), 8.63 (s, 1H). Mass Spec. M/z=567.

EXAMPLE 23 Synthesis of BIO-1198

A. To a solution of phosgene in CH₂Cl₂ at 0° C. was added a solution ofmorpholine and triethylamine in CH₂Cl₂ dropwise. The reaction was thenstirred at room temperature for 30 min and concentrated in vacuo to givea white solid. This crude product was dissolved in CH₂Cl₂ and4-aminophenylacetic acid t-butyl ester was added. The mixture wasstirred at room temperature overnight, diluted with ethyl acetate (20mL), washed with 5% citric acid (2×), sat. aq NaHCO₃ (2×) and brine(1×), dried (Na₂SO₄), filtered and concentrated to give morpholineureat-butyl ester 1198-1.

¹H-NMR (300 MHz, CDCl₃) for t-butyl ester (A): 1.40 (s, 9H), 3.38-3.46(m, 4H), 3.60-3.70 (m, 6H), 6.67 (s, 1H), 7.13 (d, 2H), 7.27 (d, 2H).

B. The morpholineurea t-butyl ester 1198-1 was dissolved in CH₂Cl₂ andtrifluoroacetic acid was added. The solution was stirred at roomtemperature for 3 h. and concentrated to give 26 mg of the correspondingcarboxylic acid 1198-2.

C. The method described in Example 1A was performed using carboxylicacid 1198-2 (26 mg) dissolved in DMF, HOBT, EDC, and the amine preparedin Example 14A to give 27 mg of crude methyl ester 1198-3.

D. A solution of crude methyl ester 1198-3 was treated as described inExample 14C to give Bio-1198. ¹H-NMR (300 MHz, DMSO-d6) for BIO 1198:0.75 (d, 3H), 0.82 (d, 3H), 1.27-1.50 (m, 3H), 2.53-2.70 (m, 2H),3.28-3.45 (m, 6H), 3.55-3.60 (m, 4H), 4.27 (m, 1H), 5.07 (bq, 1H), 5.96(bs, 2H), 6.72 (bd, 1H), 6.82 (d, 1H), 6.85 (bs, 1H), 7.09 (d, 2H), 7.35(d, 2H), 8.08 (d, 1H), 8.42 (d, 1H), 8.47 (s, 1 H). Mass Spec. M/z=569

EXAMPLE 24 Synthesis of Bio-1190

A. Amine β-5 was coupled with BocLeuOSu as described in Procedure C.This material was subjected to the conditions of Procedure D1 to givethe desired amine salt.

B. The protocol described in Example 1A was performed using2-methylphenylureaphenylacetic acid (135 mg, 0.47 mmol) in DMF (2.5 ml),HOBt (135 mg, 0.88 mmol), EDC (0.71 mmol) and the amine salt fromExample 29A (200 mg, 0.46 mmol) (treated with Et₃N until pH 10 wasreached) to give 1190-1 (235 mg, 89%) as a white solid.

C. To a stirred solution of 1190-1 (20 mg, 0.034 mmol) in MeOH (3 mL)was added aqueous LiOH (3 mL of 2N). After stirring at room temperatureovernight, the reaction mixture was cooled to 0° C. and acidified byadding TFA until pH=3-4 (pH paper). The desired product was isolated andpurified by LC (Vydac C18 column; gradient 8) to give 10 mg (0.017 mmol;50%) of BIO-1190 as a white solid. ¹H NMR (DMSO-d⁶, 300 MHz, ppm) 8.95(s, 1 H, NH), 8.39 (d, 1 H, J=9 Hz, NH), 8.11 (d, 1 H, J=9 Hz, NH), 7.88(s, 1 H, NH), 7.83 (d, 1 H, J=8 Hz, Ar), 7.36 (d, 2 H, J=8.4 Hz, Ar),7.2-7.1 (comp, 6 H, Ar), 6.92 (m, 1 H, Ar), 6.83 (d, 2 H, J=9 Hz, Ar),5.08 (m, 1 H), 4.28 (m, 1 H), 3.70 (s, 3 H, OMe), 3.39 (d, 1 H, J=8 Hz),3.31 (d, 1 H, J=7 Hz), 2.63 (m, 1 H), 2.23 (s, 3 H, Me), 1.50-1.25(comp, 3 H), 0.81 (d, 3 H, J=6 Hz), 0.75 (d, 3 H, J=6 Hz); FABMS, m/z575 (C₃₂H₃₈N₄O₆ of M⁺1 requires 575).

EXAMPLE 25 Synthesis of Bio-1197

A. Amine β-1 (0.884 g, 4.0 mmol) was coupled with BocLeuOSu (1.32 g, 4.0mmol) as described in Procedure C. This material was subjected to theconditions of Procedure D1 to give the desired amine salt (1.42 g, 85%)as a white solid.

¹H NMR (CDCl₃, 300 MHz, ppm): 7.31-7.22 (m, 5H), 7.14 (d, 1H), 5.37-5.30(m, 1H), 4.84 (m, 1H), 4.10 (m, 1H), 2.85-2.66 (m, 2H), 1.72-1.58 (m,2H), 1.51-1.49 (m,1H), 1.48 (s, 9H), 1.29 (s, 9H), 0.91 (m, 9H).

B. The procedure of Example 1A was performed using2-methylphenylureaphenylacetic acid (34 mg, 0.12 mmol), HOBT (20 mg,0.14 mmol), EDC (26 mg, 0.134 mmol) and the amine salt of Example 25A(30 mg, 0.079 mmol) in the presence of Et₃N to give 15 mg (0.028 mmol;35%) of Bio-1197 as a white foam: FABMS, m/z 545 (C₃₁H₃₈N₄O₅ of M⁺1requires 545).

EXAMPLE 26 Synthesis of BIO-1201

A. The procedure of Example 15D was performed using the free amine fromExample 15C (40 mg, 0.086 mmol) and 2-nitrophenyl isocyanate (28 mg,0.172 mmol) to give 50 mg (92%) of 1201-1 as a light yellow oil. ¹H NMR(DMSO-d⁶ ₁ 300 MHz, ppm) 8.55 (d, 1 H, NH), 8.50 (d, 1 H, NH), 8.15 (d,1 H, NH), 8.05 (d, 1 H, NH), 7.6-6.7 (11 H, Ar), 5.85 (bs, 2 H), 5.25(m, 1 H), 4.6 (m, 1 H), 3.8-3.55 (comp), 3.5 (s, 3 H, OMe), 2.75 (m, 2H), 1.7-1.4 (comp, 3 H), 0.85 (m, 6 H).

B. The procedure of Example 24C was performed using 1201-1 (50 mg, 0.079mmol) to give 17 mg (0.027 mmol; 35%) of BIO-1201 as a light yellowsolid. FABMS, m/z 620 (C₃₁H₃₅N₅O₉ of M⁺1 requires 620).

EXAMPLE 27 Syntheis of Bio-1217

A. Amine β-4 (30 mg, 0.1 mmol) was coupled withNα-t-Boc-Nε-CBZ-_(L)-Lysine-N-Hydroxysuccinimide (50 mg, 0.1 mmol) asdescribed in Example 25A to give 60 mg (93%) of 1217-1 as a white foam.¹H NMR (CDCl₃, 300 MHz, ppm) 7.35-7.25 (comp, 5 H, Ar), 6.8-6.7 (comp, 3H, Ar), 5.3-5.1 (comp, 2 H), 4.95 (m, 1 H), 4.05 (m, 1 H), 3.8 (s, 3 H,OMe), 3.78 (s, 3 H, OMe), 3.1 (m, 2 H), 2.7 (m, 2 H), 1.9-1.4 (comp),1.35 (s, 9 H, Bu^(t)), 1.3 (s, 9 H, Bu^(t))

B. Compound 1217-1 (60 mg, 0.09 mmol) in CH₂Cl₂ (5 mL) was deprotectedwith trifluoroacetic acid (0.5 mL) as described in Procedure D1 to give56 mg (100%) of 1217-2 as a white foam.

¹H NMR (CDCl₃, 300 MHz, ppm) 8.75 (bs), 7.35-7.15 (comp, Ar), 6.85-6.65(comp, Ar), 5.4 (m), 5.2-4.9 (bs, Bn), 4.15 (m), 3.75 (bs), 3.15-2.6(comp), 1.8 (m),1.4-1.0 (comp).

C. The procedure of Example 1A was performed using2-methylphenylureaphenylacetic acid (40 mg, 0.14 mmol), HOBT (23mg,0.167), EDC (30 mg, 0.158 mmol) and amine 1217-2 (56 mg, 0.093 mmol)was added in the presence of Et₃N to give 21 mg (30%) of BIO-1217 as awhite foam. ¹H NMR (DMSO-d₆, 300 MHz, ppm) 9.05 (m, 1H, NH), 8.4 (m, 1H, NH), 8.1 (m, 1 H, NH), 8.0 (m, 1 H, NH), 7.4-6.7 (comp, Ar), 5.1 (m,1 H), 5.0 (bs, 2 H), 4.2 (m, 1 H), 3.7 (bs, 6 H, OMe), 2.9-2.6 (comp),2.2 (s, 3 H, Me),1.6-1.1 (comp); FABMS, m/z 754 (C₄₁H₄₇N₅O₉ of M⁺1requires 754).

EXAMPLE 28 Synthesis of BIO-1225

A. Amine β-3 (90 mg, 0.4 mmol) coupled withNα-t-Boc-Nε-CBZ-_(L)-Lysine-N-Hydroxysuccinimide (193 mg, 0.4 mmol) asdescribed in Example 25A to give 220 mg (94%) of 1225-1 as a white foam.¹H NMR (CDCl₃, 300 MHz, ppm) 7.4-7.25 (5 H, Ar), 7.1 (m, 1 H, NH),6.8-6.65 (3 H, Ar), 5.9 (s, 2 H), 5.25 (m, 1 H), 5.15 (m, NH), 5.05 (s,2 H), 4.85 (m, 1 H), 4.0 (m, 1 H), 3.6 (s, 3 H, OMe), 3.15 (m, 2 H),2.80 (m, 2 H), 1.90-1.20 (6 H), 1.4 (s, 9 H).

B. The BOC protecting group of 1225-1 (170 mg, 0.29 mmol) was removed asdescribed in Procedure D1 to give 100 mg (71%) of free amine 1225-2 as awhite foam.

¹H NMR (CDCl₃, 300 MHz, ppm) 8.07 (d, 1 H, J=9 Hz), 7.4-7.2 (comp, 5 H),6.80-6.65 (comp, 3 H), 5.90 (s, 2 H), 5.25 (m, 1 H), 5.05 (s, 2 H), 4.98(bs, 1 H), 3.58 (s, 3 H, OMe), 3.32 (m, 1 H), 3.16 (m, 2 H), 2.27 (m, 2H), 1.90-1.70 (comp, 3 H), 1.6-1.25 (comp, 5 H).

C. The procedure of Example 1A was performed using2-methylphenylureaphenylacetic acid (44 mg, 0.155 mmol), HOBT (36 mg,0.264 mmol), EDC (47 mg, 0.248 mmol) and free amine 1225-2 (50 mg, 0.103mmol) to give 46 mg (80%) of BIO-1225-3 as a white foam. ¹H NMR(DMSO-d⁶, 300 MHz, ppm) δ 9.0-6.7 (21 H, Ar & NH),5.96 (s, 2 H), 5.1 (m,2 H), 4.98 (s, 2 H), 4.2 (m, 1 H), 3.50 (s, 3 H, OMe), 3.48-3.4 (comp, 2H), 2.88 (m, 2 H), 2.71 (m, 2 H), 2.24 (s, 3H, Me),1.6-1.0 (comp, 6 H);FABMS, m/z 752 (C₄₁H₄₅N₅O₉ of M+1 requires 752).

D. BIO-1225-3 (25 mg, 0.033 mmol) was treated as described in Example24C to give 15 mg (62%) of BIO-1225 as a white solid. FABMS, m/z 738(C₄₀H₄₃N₅O₉ of M⁺1 requires 738).

EXAMPLE 29 Synthesis of BIO-1036

A. The method described in Procedure C was followed using methyl3-amino-5-indanyl-1-propanoate (ester M-1, preparation described inProcedure B) (85 mg, 0.33 mmol) to give 1036-1 as a yellow foam (96 mg,0.22 mmol, 67%) which was used without further purification in the nextstep. ¹HNMR (CDCl₃): δ 7.15 (3H), 6.95 (1H), 5.30 (1H), 4.95 (1H), 4.15(1H), 3.55 (3H), 2.90-2.80 (6H), 2.05 (3H), 1.70 (2H), 1.35 (9H), 0.85(6H).

B. Compound 1036-1 (98 mg, 0.22 mmol) was treated as described inProcedure D to produce the corresponding amine salt. The methoddescribed in Example 1A was performed using phenylacetic acid and theresulting amine salt (in the presence of TEA) to give 1036-2 as ayellowish solid (75 mg, 0.17 mmol, 77%), which was used without furtherpurification in the next step. ¹HNMR(CDCl₃): δ 7.35-6.8 (9H), 6.25 (1H),5.25 (1H), 4.45 (1H) 3.6 (1.5H) 3.5 (1.5H), 2.80-2.60 (6H), 2.00 (2H),1.70-1.30 (5H), 0.85 (6H).

C. Using the general procedure above a small portion of compound 1036-2was hydrolyzed as described in Example 1B, purified by HPLC and theclean fractions collected to afford Bio-1036A (^(˜)2 mg) m/z=437 (98%pure by HPLC) along with Bio-1036B (^(˜)2 mg) m/z=437 (98% pure by HPLC)as white solids.

Bio-1036A: ¹H NMR (300 MHz, DMSO-d₆): δ 8.45 (1H, d, J=7.3 Hz), 8.21(1H, d, J=7.3 Hz), 7.37-7.05 (8H, m), 5.20 (1H, m), 4.37 (1H, m),3.57-3.43 (2H, m), 2.86 (4H, m), 2.69 (2H, m), 2.03 (2H, m), 1.60 (1H,m), 1.49 (2H, m), 0.91 (3H, d, J=6.3 Hz), 0.84 (3H, d, J=6.3 Hz).

Bio-1036B: ¹H NMR (300 MHz, DMSO-d₆): δ 8.45 (1H, d. J=8.4 Hz), 8.22(1H, d, J=8.4 Hz), 7.40-7.00 (8H, m), 5.18 (1H, m), 4.35 (1H, m), 3.55(2H, m), 2.85 (4H, m), 2.57 (2H, m) 2.05 (2H, m), 1.55 (1H, m), 1.40(2H, m), 0.90 (3H, d, J=6.3 Hz), 0.75 (3H, d, J=6.3 Hz).

EXAMPLE 30 Synthesis of BIO-1137

A. The method described in Procedure C was followed using methyl3-amino-3-(2-nitrophenyl)-1-propanoate (ester M-3, preparation describedin Procedure B) (58 mg, 0.22 mmol) to afford 1137-1 (106 mg, 0.22 mmol,100%) as a thick pale yellow oil.

¹HNMR(CDCl₃): δ 7.95 (1H), 7.85-7.35 (5H), 5.85 (1H), 4.95 (1H), 4.15(1H), 3.55 (1.5H), 3.50 (1.5H), 2.90 (2H), 1.70-1.60 (2H), 1.45 (9H),0.90 (6H).

B. Compound 1137-1 (106 mg, 0.22 mmol) was treated as described inExample 29B to afford 1137-2 (69 mg, 0.16 mmol, 73%) as a yellowsemi-solid.

¹HNMR(CDCl₃): δ 7.90-7.15 (10H), 6.35 (0.5H), 6.20 (0.5H), 5.75 (1H),4.45 (1H), 3.55 (1.5H), 3.50 (1.5H), 2.85 (4H), 1.70-1.30 (3H), 0.70(6H).

C. A small portion of compound 1137-1 was hydrolyzed as described inExample 1B, purified by HPLC and the clean fractions isolated to affordBio-1037A (^(˜)1 mg) m/z=442 (97% pure by HPLC) and Bio-1037B (^(˜)2 mg)m/z=442 (100% pure by HPLC).

EXAMPLE 31 Synthesis of BIO-1043

A. The commercially available N-BOC-1-aminocyclopropane carboxylic acid(80 mg, 0.4 mmol) in DMF (3 mL) was activated at room temperature usingBOP (221 mg, 0.5 mmol). After 15 minutes the methyl3-amino-3-phenyl-1-propanoate HCl salt (86 mg, 0.4 mmol) (neutralizedwith excess Hunig's base (0.15 mL, 0.8 mmol)) was added in DMF (1 mL).After stirring overnight at room temperature the reaction was dilutedwith ethyl acetate (10 mL), washed with 60% sat. bicarbonate (2×10 mL),5% citric acid (2×5 mL) and brine (10 mL), dried over sodium sulfate andconcentrated to afford 1043-1 as a white foam (143 mg, 0.4 mmol, 100%).¹H NMR (CDCl₃): δ 7.6 (1H), 7.2 (5H), 5.4-5.3 (2H), 3.55 (3H), 2.85-2.70(2H), 1.55 (2H), 1.40 (9H), 0.9 (2H).

B. A small portion of compound 1043-1 was hydrolysed as described inExample 1B and purified by HPLC. Collection of the pure fractionsafforded Bio-1043 (^(˜)3 mg) m/z=349 (100% pure by HPLC) as a whitesolid which was submitted for bioassay.

¹H NMR (300 MHz, DMSO-d₆): δ 8.55-8.05 (2H, bm), 7.5-7.15(5H, m), 5.40(2H, bm), 3.0-2.65 (2H, m), 1.45 (9H, s) 1.43-1.10 (2H, m), 0.97 (1H,bm), 0.85 (1H, bm).

EXAMPLE 32 Synthesis of BIO-1115

A. The method described in Procedure C was followed using methyl3-amino-3-(4-chlorophenyl)-1-propanoate HCl salt (ester M-1, preparationdescribed in procedure B) (68 mg, 0.27 mmol) to afford 1115-1 (94 mg,0.22 mmol, 82%) as a white foam.

¹HNMR (CDCl₃): δ 7.35 (1H), 7.25-7.10 (4H), 5.35 (1H), 4.95 (1H), 4.05(1H), 3.60 (1.5H), 3.55 (1.5H), 2.80-2.65 (2H), 1.65 (2H), 1.40 (10H),0.80 (6H).

B. Compound 1115-1 (68 mg, 0.27 mmol) was treated as described inExample 29B to afford crude 1115-2 (67 mg, 0.15 mmol, 68%) as a paleyellow solid.

¹H NMR: δ 7.50 (1H), 7.40-7.00 (9H), 6.20 (1H), 5.25 (1H), 4.45 (1H),3.60 (1.5H), 3.55 (1.5H), 2.7-2.55 (4H), 1.65-1.40 (3H), 0.80 (6H).

C. A small portion of crude 1115-1 was hydrolysed, purified by LC andthe pure fractions collected to afford Bio-1115A (^(˜)1 mg) m/z=431(100% pure by HPLC) along with Bio-1115B (^(˜)2 mg) m/z=431 (100% pureby HPLC) as white solids.

Bio-1115A: ¹H NMR (300 MHz, DMSO-d₆): δ 8.46 (1H, d, J=8.2 Hz), 8.27(1H, d, J=8.2 Hz), 7.46-7.18 (9H, m), 5.20 (1H, m), 4.35 (1H,m),3.60-3.45 (2H, m), 2.71 (2H, d, J=7.3 Hz), 1.63 (1H, m), 1.48 (2H, m),0.91 (3H, d, J=6.4 Hz), 0.84 (3H, d, J=6.4 Hz).

Bio-1115B: ¹H NMR (300 MHz, DMSO-d₆): δ 8.60 (1H, d, J=8 Hz), 8.26 (1H,d, J=8 Hz), 7.45-7.15 (9H, m), 5.18 (1H, m), 4.35 (1H, m), 3.50 (2H, m),2.70 (2H, m), 1.50(1H, m), 1.42 (2H, m), 0.85 (3H, d, J=6.3 Hz), 0.75(3H, d, J=6.3 Hz).

EXAMPLE 33 Synthesis of BIO-1129

A. To a solution of 4-(phenylurea)phenylacetic acid (540 mg, 2.0 mmol;prepared in Example 22C) in DMF (5 mL) was added EDC (460 mg., 2.4mmol). After storing at room temperature for 15 min, phenylalaninet-butyl ester HCl salt (515 mg, 2.0 mmol) which was neutralized withexcess Hunig's base (0.7 mL, 4.0 mmol) was added in DMF (3 mL). Afterstirring overnight the reaction was diluted with ethyl acetate (20 mL)and washed with 60% sat. bicarbonate (2×10 mL), citric acid (2×10 mL),brine (2×10 mL), dried over sodium sulfate and concentrated to affordcrude 1129-1 (662 mg, 1.40 mmol, 70%) as a thick pale yellow oil.

¹HNMR (CDCl₃): δ 7.45-6.90 (16H), 6.45 (1H), 4.70 (1H), 3.4 (2H),3.15-2.90 (2H), 1.35 (9H).

B. To crude product 1129-1 (662 mg, 1.40 mmol) was added methylenechloride (5 mL) followed by TFA (1 mL). After stirring overnight thereaction was concentrated to dryness and dried on a vacuum pump. A smallportion (21 mg, 0.05 mmol) was dissolved in DMF (1 mL) and HOBt (11 mg,0.07 mmol) was added followed by EDC (14 mg, 0.06 mmol). After stirringfor 15 min at room temperature amine β-3 (13 mg, 0.05 mmol) was added inDMF (0.5 mL). After stirring overnight the reaction was diluted withethyl acetate (20 mL), washed with sat. bicarbonate (2×10 mL), citricacid (10 mL), brine (10 mL) dried over sodium sulfate and concentratedto afford crude 1129-2 (26 mg, 0.04 mmol, 80%) as a light tan solid.¹HNMR (CDCl₃): δ 8.4 (1H), 7.4-6.5 (19H), 5.95 (2H), 5.7 (1H), 5.25(1H), 4.70 (1H), 3.65-3.50 (5H), 3.10-2.65 (4H).

C. A small aliquot of crude 1129-2 was hydrolysed as described inExample 1B and purified by HPLC to afford:

Bio-1129A (^(˜)1.5 mg) m/z=609 (80:20 ds) (100% pure by HPLC); and

Bio-1129B (^(˜)2 mg) m/z=609 (9:91 ds) (100% pure by HPLC) as whitesolids.

Bio-1129A: ¹H NMR (300 MHz, DMSO-d₆): δ 8.18 (1H, s), 8.14 (1H, s), 8.50(1H, bd), 8.23 (1H, bd), 7.50 (2H, d, J=8.1 Hz), 7.40-7.10 (9H, m),7.08-6.72 (6H, m), 6.04 (2H, s), 5.15 (1H, m), 4.07 (1H, m), 3.38 (2H,m, 3.05-2.70 (2H, m), 2.62 (2H, s).

EXAMPLE 34 Synthesis of BIO-1131

A. The method of Example 1A was performed using phenylureaphenyl aceticacid (prepared in Example 22C) and isoleucine methyl ester HCl salt (362mg, 2.0 mmol) (treated with TEA) to afford crude 1131-1 (344 mg, 1.0mmol, 51) as a clear thick oil.

¹HNMR (CDCl₃): δ 7.7 (1H), 7.35-6.95 (10H), 6.60 (1H) 4.55 (1H), 3.65(3H), 3.45 (2H), 1.90 (1H), 1.45-1.20 (3H), 0.85 (5H).

B. To a solution of crude 1131-1 (344 mg, 0.95 mmol) in methanol (5 mL)was added 2N LiOH (2 mL). After stirring overnight the methanol wasremoved, H₂O (5 mL) added and the pH adjusted to pH=1-2. The aqueouslayer was extracted with ethyl acetate (5×20 mL) dried over sodiumsulfate and concentrated to give 1131-2 (365 mg, 0.95 mmol, 100%) as atan solid.

¹H NMR (CDCl₃): δ 8.70 (2H), 8.30 (1H), 7.60-7.20 (8H), 7.00 (1H), 4.25(1H), 3.55 (2H), 1.90 (1H), 1.55 (1H), 1.30 (2H), 0.85 (5H).

C. Prepared from 1131-2 (27 mg, 0.07 mmol) and amine β-3 (11 mg, 0.07mmol) as described in Example 1A to afford crude 1131-3 (34 mg, 89%), asa pale brownish solid. ¹H NMR (CDCl₃): δ 8.3 (2H), 7.45-6.65 (16H), 5.45(1H), 4.45-4.30 (1H), 3.55 (2H), 3.2-2.90 (2H), 2.00-0.70 (9H).

D. A small aliquot of crude 1131-3 was hydrolysed as described inExample 1B and purified by HPLC to afford Bio-1131A (^(˜)2 mg)m/z=531(100:0 ds) (100% pure by HPLC)and Bio-1131B (^(˜)3 mg) m/z=531 (0:100ds) (100% pure by HPLC) as white solids.

Bio-1131A: ¹H NMR (300 MHz, DMSO-d₆): δ 8.69 (1H, s), 8.63 (1H, s), 8.50(1H, d, J=8.1 Hz), 7.50 (2H, d, J=7.8H₂), 7.44-7.22 (8H, m), 7.19 (2H,d,J=8.4 Hz), 7.00 (1H, m), 5.27 (1H, m), 4.36 (1H, m), 3.52 (2H, m),3.00 (2H, bm), 2.71 (2H, d, J=7.3H_(z)), 1.70 (1H, bm), 1.44-1.26 (1H,m), 1.22-1.00 (3H, m), 0.95-0.78 (5H, m).

Bio-1131B: ¹H NMR (300 MHz, DMSO-d₆): δ 8.73 (1H, s), 8.68 (1H, s), 8.60(1H, d, J=8 Hz), 8.15 (1H, d, J=8 Hz), 7.50 (2H, d, J=7.9 Hz), 7.42 (2H,d, J=8.4 Hz), 7.37-7.23 (5H, m), 7.20 (2H, d, J=8.4 Hz), 7.00 (1H, m),5.35 (1H, m), 4.23 (1H, m), 3.50 (2H, m), 3.05 (2H, bm), 2.71 (2H, m),1.72 (1H, bm), 1.20 (3H, m), 0.72-0.60 (5H, m).

EXAMPLE 35 Synthesis of BIO-1136

A. The method described in Example 1A was performed utilizingcommercially available N-BOC-S-benzyl-cysteine (25 mg, 0.08 mmol) andmethyl 3-amino-3-phenyl-1-propanoate (17 mg, 0.09 mmol) to afford crudeprotected amine 1136-1 (42 mg, 0.08 mmol, 100%).

¹H NMR (CDCl₃): δ 7.35 (10H), 5.40-5.20 (2H), 4.20 (1H), 3.65 (1.5H),3.55 (1.5H), 3.54 (1.5H), 3.25-2.65 (6H), 1.45-1.30 (9H).

B. The protected amine 1136-1 was treated as described in Procedure D togive the TFA-amine salt 1136-2.

C. The method described in Example 22D was performed utilizing freeamine 1136-2 (42 mg, 0.08 mmol) (TEA treatment) to afford crude 1136-3which was used in the hydrolysis step without further purification.

D. A small aliquot of crude 1136-3 was hydrolysed as described inExample 1B and purified by HPLC to afford Bio-1136 (^(˜)4 mg)m/z=611(100% pure by HPLC) as a white solid. ¹H NMR (300 MHz, DMSO-d₆): δ9.05(2H, bm), 8.90 (1H, br), 8.37(1H, br), 7.50 (1H, d, J=7.7 Hz), 7.45 (1H,d, J=8.3 Hz), 7.4-7.2 (9H, m), 7.00 (1H, m) 5.25 (1H, br), 4.65 (1H,br), 3.5-3.2 (4H, m), 2.70 (2H, bm)

EXAMPLE 36 Synthesis of BIO-1176

A. To a solution of commercially available N-BOC-aspartic acid α-benzylester (500 mg, 1.55 mmol) in DMF (5 mL) was added HOBt (283 mg, 2.10mmol) followed by EDC (343 mg, 1.80 mmol). After stirring for 15 minutesat room temperature thiomorpholine (500 mg, 1.54 mmol) was addedfollowed by Hunig's base (0.7 mL, 92 mmol) and the reaction mixturestirred at room temperature overnight. The reaction was worked up bydiluting with ethyl acetate (25 mL) and washing with 60% sat.bicarbonate (5 mL), 5% citric acid (5 mL) and brine (5 mL). The organicswere separated, dried over sodium sulfate and concentrated to give ester1176-1 as a thick orange oil (421 mg, 1.03 mmol, 69%). ¹H NMR (CDCl₃): δ7.13 (5H, m), 5.69 (1H, bd, J=9.4 Hz) 5.03 (1H, d, J=12.6H^(Z)), 4.42(1H, m), 3.61 (1H, m), 3.60-3.40 (4H, m), 2.96 (1H, bm), 2.58 (1H, bm),2.35 (4H, m) 1.22 (9H, s)

B. Ester 1176-1 (100 mg, 0.25 mmol) was treated as described in Example1B to afford acid 1176-2 (76 mg, 0.24 mmol, 96%) as a clear thick oil.

¹H NMR (CDCl₃): δ 7.39-7.28 (5H, m), 7.15-6.70 (1H, br), 5.70 (1H, bs,J=6.3 Hz), 4.55 (1H, br), 4.40-3.40 (4H, m) 3.15 (1H, m), 2.80-2.52 (5H,m), 1.43 (9H, s).

C. The method of Example 1A was performed using acid 1176-2 (32 mg, 0.10mmol), in DMF, HOBT, EDC, and amine β-3 to afford 1176-3 (36 mg, 0.07mmol, 70%) as a thick pale yellow oil. ¹H NMR (CDCl₃): 67 7.71 (1H, br),6.61 (3H, m) 6.00 (0.5H, br), 5.90 (1H, s), 5.77 (0.5H, br), 5.21 (1H,m), 4.51 (1H, bm), 3.90-3.40 (4H, m), 3.39 (3H, s), 3.12-3.00 (1H, m),2.85-2.65 (3H, m), 2.63-2.45 (4H, m), 1.43 (4.5H, s), 1.43 (4.5H, s).

D. The protected amine 1176-3 (36 mg, 0.07 mmol) was treated asdescribed in Procedure D to give TFA-amine salt 1176-4 (51 mg, 0.07mmol, 100%) as a pale yellow solid.

E. The method described in Example 22D was performed utilizing freeamine 1176-4 (42 mg, 0.08 mmol) (after TEA treatment) to afford crude1176-5 which was used in the hydrolysis step without furtherpurification. ¹H NMR (CDCl₃): δ 7.95-6.9 (13H, m), 6.61 (3H, s), 5.85(2H, s) 5.23 (1H, m), 4.88 (1H, m), 3.89-3.60 (4H, s), 3.55 (3H, s),3.43 (2H, br), 3.11-2.96 (2H, m), 2.71 (2H, m), 2.46 (4H, m).

F. Crude 1176-5 was hydrolyzed as described in Example 1B and injectionof a small aliquot into the HPLC afforded Bio-1176 (˜4 mg) m/z=662 (>99%pure by HPLC) as a white solid. ¹HNMR: (DMSO-d₆) δ 8.69 (2H, d, J=9.8Hz), 8.33 (1H, d, J=8.0 Hz), 8.26 (1H, d, J=8.0 Hz), 7.61 (2H, d, J=8.0Hz), 7.43 (2H, d, J=8.0 Hz), 7.34 (2H, m), 7.21 (2H, d. J=8.0 Hz),7.10-6.95 (4H, m), 6.11(2H, s), 5.13(1H, m), 4.68(1H, m), 3.71(4H, br),3.56-3.18 (2H, m), 2.73-2.46 (8H, m).

EXAMPLE 37 Synthesis of BIO-1177

A. The procedure described in Example 36A was carried out usingmethylpropargylamine in place of thiomorpholine to afford crude 1177-1(374 mg, 0.99 mmol, 66%) as a white foam. ¹H NMR (CDCl₃): δ 7.20 (5H),5.25 (1H), 5.10 (2H), 4.45 (1H), 4.15-3.8 (2H), 3.15-2.65 (5H), 2.2-2.15(1H), 1.30 (9H).

B. Crude 1177-1 was treated as described in Example 1B to afford acid1177-2 (76 mg, 0.26 mmol, 96%) as a clear oil. ¹H NMR (CDCl₃):δ5.35(1H), 4.55(1H), 4.35-3.8(2H), 3.30-2.65(5H), 2.4-2.25(1H),1.45(9H).

C. The method of Example 1A was performed using acid 1177-2 (76 mg, 0.26mmol), in DMF, HOBT, EDC, and amine β-3 to afford crude 1177-3 (78 mg,0.15 mmol) as a white foam. ¹H NMR (CDCl₃): δ 7.70 (1H), 7.35 (3H), 6.65(2H), 5.80 (1H) 5.30-5.00 (2H), 4.60(1H), 4.45-3.80 (2H), 3.60 (3H),3.30-2.70 (5H), 2.30 (1H), 1.45 (4.5H), 1.40 (4.5H).

D. The protected amine 1177-3 (78 mg, 0.15 mmol) was treated asdescribed in Procedure D to afford TFA-amine salt 1177-4.

E. The method described in Example 22D was performed utilizing freeamine 1177-4 to afford 1177-5 (52 mg, 0.08 mmol, 77%) as a tan solid. ¹HNMR: (CDCl₃) δ 7.5-6.9 (14H), 6.65 (3H), 5.85 (2H) 5.25-5.00 (2H), 4.85(1H), 4.25-3.70 (2H), 3.60 (3H), 3.55 (2H), 3.30-2.65 (5H), 2.22 (1H).

F. A small portion of 1177-5 was hydrolyzed as described in Example 1Bto afford Bio-1177 (^(˜)2 mg) m/z=628 (100% pure by HPLC) as a whitesolid.

¹H NMR: (DMSO-d₆) δ8.64 (2H, bd), 8.27 (2H, bm), 7.55-7.13 (7H, m),7.11-6.75 (3H, m) 6.15 (2H, s), 5.12 (1H, bm), 4.65 (1H, bm), 4.25 (2H,bm), 3.25 (2H, m), 3.05 (2H, br) 2.88 (1H, bm), 2.62 (2H, m).

EXAMPLE 38 Synthesis of BIO-1214

A. The procedure described in Example 36A was carried out on theN-BOC-aspartic acid α-benzyl ester (1.60 g, 4.9 mmol) usingdimethylamine in place of thiomorpholine to afford ester 1214-1 (1.43 g,4.1 mmol, 83%) as a thick colorless oil.

¹H NMR (CDCl₃) δ 7.32 (5H, m), 5.85 (1H, br), 5.15 (2H, m) 4.55 (1H,br), 3.12 (1H, m), 2.94 (3H, s), 2.88 (3H, s), 2.73 (1H, m), 1.40 (9H,s).

B. Ester 1214-1 (124 mg, 0.33 mmol) was dissolved in ethyl acetate (2mL) and 10% Pd/C (^(˜)50 mg) was added and the mixture was hydrogenatedunder pressure (40 psi) for 2 h. The reaction was filtered throughCelite® and concentrated to afford acid 1214-2 (95 mg, 0.33 mmol, 100%),as a colorless oil. ¹H NMR: (CDCl₃) δ 5.81 (1H, bm), 4.48 (1H, bs), 3.15(1H, m), 3.00 (3H, s), 2.93 (3H, s), 2.59 (1H, m), 1.39 (9H, s).

C. The method of Example 1A was performed using acid 1214-2 (28 mg, 0.10mmol) and amine β-3 (17 mg, 0.80 mmol) to afford protected amine 1214-3(55 mg, 0.10 mmol, 100%) as a white foam. ¹H NMR: (CDCl₃) δ7.77 (1H,bd), 6.71 (3H, m), 6.11 (1H, bd), 5.91 (2H, s) 5.25 (1H, m), 4.51 (1H,br), 3.60 (3H, s), 3.12 (1H, m), 2.94 (3H, s), 2.90 (3H, s), 2.88-2.68(2H, m), 2.48 (1H, m), 1.43 (9H, s).

D. The protected amine 1214-3 (55 mg, 0.10 mmol) was treated asdescribed in Procedure D to afford TFA-amine salt 1214-4.

E. The method described in Example 22D was performed utilizing freeamine 1214-4 to afford 1214-5 (31 mg, 0.05 mmol, 50%) as a tan solid. ¹HNMR (CDCl₃): δ 7.45-6.90 (13H, m), 6.61 (3H, m), 5.85 (2H, s), 5.24 (1H,m), 4.82 (1H, m), 3.55 (3H, s), 3.47 (2H, m), 3.08-2.94 (1H, m), 2.92(3H, s), 2.84 (3H, s), 2.77-2.50 (2H, m), 2.45 (1H, m)

F. A small portion of 1214-5 was hydrolyzed as described in Example 1Bto afford BIO-1214 (^(˜)2 mg) m/z=604 (100% purity by HPLC) as a whitesolid.

EXAMPLE 39 Synthesis of BIO-1215

A. To a solution of amide 1214-1 (prepared in Example 38A) (671 mg, 1.9mmol) in dry tetrahydrofuran (5 mL) cooled to 0° C. was added 1 NBH₃/THF solution (4.1 mL, 3.8 mmol) dropwise. After stirring thereaction mixture for 2 h at room temperature the reaction was quenchedwith methanol (2 mL) and concentrated to dryness. Methanol (5 mL) wasadded and removed three times to remove all (MeO)₃B formed. Drying underhigh vacuum afforded amine 1215-1 (623 mg, 1.7 mmol, 90%) as a thickcolorless oil. ¹H NMR (CDCl₃) δ 7.38 (5H, M), 5.48 (1H, bm), 2.65-2.35(8H, m), 1.95 (2H, m), 1.42 (9H, s).

B. Amine 1215-1 (124 mg, 0.34 mmol) was subjected to catalytichydrogenation using methanol/ethyl acetate/acetic acid as solvent and10% Pd/C (^(˜)50 mg). After 2 h the reaction mixture was filtered andconcentrated to give acid 1215-2 (90 mg, 0.33 mmol, 97%) as a thickcolorless oil.

¹H NMR (CDCl₃) δ 5.91 (1H, br), 3.95 (1H, br), 3.54 (1H, bm), 2.71-2.42(8H, m), 2.15 (2H, br), 1.33 (9H, s).

C. The method of Example 1A was performed using acid 1215-2 (55 mg, 0.12mmol) and the amine β-3 (22 mg, 0.10 mmol) to afford protected amine1215-3 (44 mg, 0.09 mmol, 90%) as a white foam. ¹H NMR(CDCl₃) δ6.75 (3H,m), 6.51 (1H, bd), 5.91 (2H, s), 5.30 (1H, m), 4.37-4.12 (2H, m), 3.61(3H, s), 2.90-2.65 (2H, m), 2.55-2.00 (10H, m), 1.42 (9H, s).

D. The protected amine 1215-3 (44 mg, 0.09 mmol) was treated asdescribed in Procedure D to afford TFA-amine salt 1215-4.

E. The method described in Example 22D was performed utilizing freeamine 1215-4 to afford 1215-5 (38 mg, 0.06 mmol, 70%) as a white solid.

¹H NMR (CDCl₃) δ 7.41-6.90 (13H, M), 6.71 (3H, m), 5.91 (2H, s), 5.29(1H, m), 4.21 (1H, m), 3.61 (3H, s), 3.45 (2H, m), 2.90-2.70 (2H, m),2.40-1.95 (10H, m).

F. A small portion of 1214-5 was hydrolyzed as described in Example 1Bto afford BIO-1215 (^(˜)3 mg) m/z=590 (100% pure by HPLC) as a whitesolid.

EXAMPLE 40 Synthesis of BIO-1227

A. The method as described in Example 1B was performed usingcommercially available BOC-S-methyl-cysteine (28 mg, 0.12 mmol) andamine β-3 (21 mg, 0.10 mmol) to afford protected amine 1227-1 (32 mg,0.07 mmol, 70%) as a white foam.

¹H NMR (CDCl₃) δ 7.38 (1H, bd), 6.81-6.67 (3H, m), 5.90 (2H, s), 5.40(1H, bd), 5.37 (1H, m), 4.20 (1H, m), 3.59 (3H, s), 2.95-2.68 (4H, m),2.10 (3H, s), 1.43 (9H, s).

B. The protected amine 1227-1 (32 mg, 0.07 mmol) was treated asdescribed in Procedure D to afford TFA-amine salt 1227-2.

C. The method described in Example 22D was performed utilizing freeamine 1227-2 and the 2-methylphenylureaphenylacetic acid (28 mg. 0.10mmol) to afford crude ester 1227-3 (29 mg, 0.047 mmol, 67%) as a lighttan solid. ¹H NMR (CDCl₃) δ 7.62 (1H, bd), 7.4-6.9 (12H, m), 6.80 (3H,m) 5.90 (2H, s) 5.15 (1H, m), 4.45 (1H, m), 3.63-3.45 (5H, m), 3.15-2.61(4H, m), 2.21 (3H, s), 2.10 (3H, s).

D. A small aliquot of crude ester 1227-3 was hydrolyzed as described inExample 1B to afford BIO-1227 (^(˜)4 mg) m/z=593 (>99% pure by HPLC) asa white solid. ¹H NMR (DMSO-d₆): δ9.01 (1H, s), 8.67 (1H, d, J=7.9 Hz),8.31 (1H, d, J=8.3 Hz), 7.97 (1H, s), 7.90 (1H, d, J=8 Hz), 7.44 (2H,d,J=8.3 Hz), 7.23 (4H, m), 6.99 (2H, m), 6.85 (2H, m), 6.03 (2H, s), 5.16(1H, m), 4.54 (1H, M), 3.39 (2H, m), 2.81-2.58 (4H, m) 2.30 (3H, s),2.05 (3H, s).

EXAMPLE 41 Synthesis of BIO-1149

A. To a solution of the product from Procedure C (272 mg, 0.67 mmol) inCH₂Cl₂ (2.5 ml) was added TFA (2.5 ml) slowly and the mixture wasstirred at room temperature for 1 h. The solvents were removed to givean oil. This oil was dissolved in CH₂Cl₂ (2.5 ml). To this solution wasadded Et₃N to pH 9 and then succinimidyl 2-quinolinecarboxylic acid (170mg, 0.63 mmol). The mixture was stirred at room temperature for 1 hfollowed by usual workup (5% citric acid, 5% NaHCO₃ and sat. NaCl) toafford ester 1149-1 (200 mg, 76%) as a white solid.

B. Acid 1149-1 (200 mg, 0.43 mmol) was dissolved in methanol (1.5 ml)and to the solution was added 1M aqueous LiOH (0.5 ml). The mixture wasstirred at room temperature for 3 h and neutralized with 5% citric acidto pH 3 and was extracted with EtOAc (3×5 ml). The combined extractswere dried (Na₂SO₄) and concentrated to afford 155 mg (82.5%) crude1149. A small amount of the crude product (30 mg) was purified by HPLCto give BIO-1149, and the diastereomers were separated. HPLC: Roomtemperature; A: 36 min; B:38 min. FAB-MS=434. ¹H NMR: (CDCl₃, 300 MHz,ppm) 8.72 (m, 1H), 8.30-7.98 (m, 3H), 7.82-7.64 (m, 2H), 7.60-7.51 (m,1H), 7.30-7.09 (m, 5H), 5.46-5.38 (m, 1H), 4.86-4.72 (m, 1H), 2.92-2.74(m, 2H), 1.88-1.61(m, 3H), 0.96-0.83 (m, 6H).

EXAMPLE 42 Synthesis of BIO-1152

A. To a solution of the product of Procedure D2 (33 mg, 0.1 mmol) inCH₂Cl₂ (0.5 ml) was added 2,2-dimethylbutyric acid chloride (14 mg, 0.1mmol) and Et₃N (50 μl). The mixture was stirred at room temperature for16 h. The usual workup (5% NaHCO₃, 5% citric acid and sat. NaCl)afforded 1152-2 (37 mg, 76%) as a white solid. ¹H NMR: (CDCl₃, 300 MHz,ppm) 7.32-7.19 (m,5H), 6.08 (s, 1H), 5.36-5.27 (m, 1H), 4.53-4.44 (m,1H), 2.86-2.61 (m, 2H), 2.05 (s, 2H), 1.26 (s, 9H), 1.01 (s, 9H),0.99-0.84 (s, 9H).

B. Ester 1152-2 was dissolved in CH₂Cl₂ (2.5 ml) and TFA (2.5 ml) andstirred at room temperature for 3 h to afford an oil. The purificationof the oil by HPLC to give a pure BIO-1152. ¹H NMR: (CDCl₃, 300 MHz,ppm) 8.29 (d, 1H), 7.44 (d, 1H), 7.34-7.18 (m, 5H), 5.44-5.32 (m, 1H),4.78-4.69 (m, 1H), 3.21-3.14 (m, 2H), 2.98-2.77 (dd, 2H), 1.59-1.38 (m,3H), 0.96 (s, 9H), 0.84 (d, 3H), 0.73 (d, 3H).

EXAMPLE 43 Synthesis of BIO-1089

A. To a solution of amine β-6 (2.2 g, 8.76 mmol) in CH₂Cl₂ (25 ml) wasadded N-BOC-methionine succinimidyl ester (2.77 g, 8.0 mmol) and Et₃N (5drops) and the mixture was stirred at room temperature for 1.5 h. Themixture was washed with 5% citric acid (2×10 ml), 5% NaHCO₃ (2×10 ml)and sat. NaCl (15 ml), dried (Na₂SO₄) and concentrated to give 1089-1(3.2 g, 83%) as a white solid. ¹H NMR: (CDCl₃, 300 MHz, ppm) 7.27 (d,2H), 6.81 (d, 2H), 5.31-5.20 (m, 2H), 4.38-4.28 (m, 1H), 3.72 (s, 3H),2.82-2.64 (m, 2H), 2.12 (s, 3H), 1.44 (s, 9H), 1.30 (s, 9H).

B. To a solution of 1089-1 (3.2 g, 6.64 mmol) in EtOAc (15 ml) was addeda 1M HCl-EtOAc solution (40 ml) and the mixture was stirred at roomtemperature for 4.5 h. The reaction mixture was quenched with H₂O (60ml) and the aqueous layer was collected. It was neutralized with solidNaHCO₃ to pH 8 and was extracted with EtOAc (2×45 ml). The combinedorganic extracts were washed with sat. NaCl (20 ml), dried (Na₂SO₄) andconcentrated to afford 1089-2 (1.7 g, 67%) as an oil.

¹H NMR: (CDCl₃, 300 MHz, ppm) 7.98 (d, 1H), 7.19 (d, 2H, J=8.3 Hz), 6.81(d, 2H, J=8.3 Hz), 5.32-5.18 (m, 1H), 3.74 (s, 3H), 3.48-3.44 (m, 1H),2.82-2.62 (m, 2H), 2.53 (t, 2H), 2.18-2.06 (m, 1H), 2.04 (s, 3H),1.8-1.66 (1H), 1.31 (s, 9H).

C. The method of Example 22D was performed using 1089-2 (1.7 g, 4.45mmol) to afford 1089-3 (2.3 g, 81.6%) as a solid. This material was usedin the next step without further purification.

¹H NMR: (DMSO-d⁶, 300 MHz, ppm) 8.60 (d, 2H), 8.41 (d, 1H), 8.24 (d,1H), 7.44 (d, 2H), 7.31(d. 2H), 7.26 (t, 2H), 7.13 (t, 2H), 6.91 (t,1H), 6.79 (d, 2H), 5.10-5.01 (m, 1H), 4.36-4.33 (m, 1H), 3.68 (s, 3H),3.29 (s, 2H), 2.61-2.58 (m, 2H), 1.89 (s, 3H), 1.26 (s, 9H).

D. Compound 1089-3 (2.3 g, 3.63 mmol) was dissolved in 4N HCl-dioxane (8ml) and the solution was stirred at room temperature for 16 h. After thedioxane was removed, ether (15 ml) was added and mixture was stirred for10 min. The precipitate was collected and was recrystallized frommethanol to give pure BIO-1089 as a pale brown solid. FAB-MS=579.

¹ NMR: (DMSO-d⁶, 300 MHz, ppm) 8.76 (d, 2H), 8.52 (d, 1H), 7.54 (d, 2H),7.46 (d, 2H), 7.36 (t, 1H), 7.34-7.26 (m, 4H), 7.04 (t, 1H), 6.95 (d,2H), 5.22-5.20 (m, 1H), 4.46-4.35 (m, 1H), 3.81 (s, 3H), 3.50, (s, 2H),3.20 (m, 2H), 2.79-2.73 (m, 2H), 2.35 (t, 2H), 2.03 (s, 3H), 1.87-1.80(m, 2H).

EXAMPLE 44 Synthesis of BIO-1090

A. The method described in Procedure C was followed using amine β-10 (28mg, 1.0 mmol) to afford 1090-1 (38 mg, 84%) as a white solid. ¹H NMR:(CDCl₃ 300 MHz, ppm) 7.08 (m, 1H), 6.82 (s, 1H), 6.74-6.70 (m, 2H),5.24-5.15 (m, 1H), 4.98-4.93 (m, 1H), 4.16-4.13 (m, 4H), 2.74-2.53 (m,2H), 1.62-1.42 (m, 3H), 1.44 (s, 9H), 1.40 (s, 9H), 0.89 (m, 6H).

B. The white solid of 1090-1 (38 mg, 0.77 mmol) was treated as describedin Procedure D1 to give 1090-2 as an oil. This compound was used in nextstep without further purification. ¹H NMR: (CDCl₃, 300 MHz, ppm)7.24-7.15 (m, 2H), 6.84-6.61 (m, 3H), 5.81-5.78 (m, 1H), 4.23 (s, 4H),4.19-4.08 (m, 1H), 2.88-2.62(m, 2H), 1.70-1.46 (m, 3H), 0.90-0.81 (m,6H).

C. The method of Example 22D was performed using amine 1090-2 to affordcrude 1090 (27 mg, 59%). The purification of crude product by HPLC gavepure BIO-1090 as a white solid. FAB-MS=603.

EXAMPLE 45 Synthesis of BIO-1194

A. To a well-stirred cold solution of methyl p-aminophenylacetate (9.8g, 59.4 mmol) in CH₂Cl₂ (200 ml) and Et₃N (25 ml, 18 g, 178.2 mmol) wasadded COCl₂ (96 ml of 1.9M solution in toluene) through an additionalfunnel for 1 h. The reaction mixture was stirred at 0° C. for another 1h. The reaction mixture was concentrated and ether:pet ether (3:1) (125ml) was added. The solid was filtered and the filtrate was collected.Removal of the solvents gave crude 1194-1 as a brown liquid. Thepurification of crude product by distillation (118-120° C./10 mm) gavepure 1194-1 (8.5 g, 75%) as a colorless liquid.

¹H NMR: (CDCl₃, 300 MHz, ppm) 7.20 (d, J=8.4 Hz), 7.02 (d, J=8.4 Hz),3.69 (s, 3H), 3.48 (s, 2H).

B. To a solution of 1194-1 (5.73 g, 30.0 mmol) in CH₂Cl₂ (60 ml) wasadded 2-aminopyridine (2.82 g, 30 mmol) in portions. The mixture wasstirred at room temperature for 0.5 h then 35° C. for 0.5 h. Theresulting mixture was diluted with pet ether (60 ml) and a white solidwas formed. Filtration of the solid gave pure 1194-2 (8.35 g, 98%) as awhite solid.

¹H NMR: (CDCl₃, 300 MHz, ppm): 8.20 (s, 2H), 7.62-7.51 (m, 3H), 7.33 (d,2H), 7.01 (d, 2H), 6.89-6.85 (m, 1H), 3.70 (s, 3H), 3.59 (s, 2H).

C. Compound 1194-2 (5.7 g, 20.0 mmol) was dissolved in methanol (20 ml)and to this was added 1N NaOH (40 ml). The mixture was heated until aclear solution was formed and was stirred at room temperature for 16 h,followed by a careful neutralization with 1N HCl to pH 7 then withacetic acid to pH 3. The white solid thus formed was filtered and washedwith methanol (15 ml) and ether (2×30 ml) to give 1194-3 (4.7 g, 87%) asa white powder. ¹H NMR: (DMSO-D⁶, 300 MHz, ppm) 10.62 (br, s, 1H), 9.53(br, s, 1H), 8.39 (d, 1H), 7.82 (t, 1H), 7.63-7.55 (m, 1H), 7.33-7.27(d, 2H), 7.14-7.08 (m, 1H), 3.62 (s, 3H).

D. Standard Procedure C was followed to prepare 1194-4 by coupling amineβ-6 (2.65 g, 10.56 mmol) with BocLeuOSu (3.28 g, 10 mmol) in CH₂Cl₂ (25ml) and Et₃N (5 drops) then followed by deprotection (TFA/CH₂Cl₂) toafford 1194-4 (4.5 g, 83.6%) in two steps 1194-4-Boc: ¹H NMR: (CDCl₃,300 MHz, ppm) 7.18 (d, 2H), 6.36 (d, 2H), 5.13-5.10 (m, 1H), 4.12-4.01(m, 1H), 3.72 (s, 3H), 2.79-2.60 (m, 2H), 1.62-1.40 (3H), 1.38 (s, 9H),1.26 (s, 9H), 0.85-0.80 (m, 6H). 1194-4: ¹H NMR: (CDCl₃, 300 MHz, ppm)7.10 (d, 2H), 6.78 (d, 2H), 5.43-5.27 (m, 1H), 4.21-4.06 (m, 1H), 3.71(s, 3H), 2.95-2.76 (m, 1H), 2.75-2.56 (m, 1H), 1.62-1.32 (m, 6H).

E. The method of Example 1A was followed using acid 1194-3 (1.36 g, 5.0mmol) and amine 1194-4 to afford crude BIO-1194 (2.1 g, 78%) as a whitesolid. The pure product (purity >97.5%) was obtained by crystallizationfrom methanol. ¹H NMR: (CDCl₃, 300 MHz, ppm) 8.03-7.97(m, 2H), 7.59 (m,1H), 7.51 (d, 2H), 7.18-7.07 (m, 4H), 6.27 (d, 2H), 5.24 (m, 1H),4.39-4.36 (m, 1H), 3.61 (s, 3H), 3.43 (s, 2H), 2.69-2.66 (m, 2H),1.54-1.33 (m, 2H), 0.86-0.75 (m, 6H).

EXAMPLE 46 Synthesis of BIO-1180

A. The method described in Example 45A was followed using t-butylp-aminophenylacetate to give 1180-1 in 94% yield. FAB-MS=234. ¹HNMR:(CDCl₃, 300 MHZ, ppm) 7.18 (d, 2H, J=8.2 Hz), 6.98 (d, 2H, 8.2 Hz),3.49 (s, 3H), 1.45 (s, 9H).

B. To a solution of isocyanate 1180-1 (233 mg, 1.0 mmol) in CH₂Cl₂ (5ml) was added 2-aminothiazole (100 mg, 1.0 mmol) and the mixture washeated until a clear solution was formed and was stirred at roomtemperature for 1 h. Removal of the solvents gave 1180-2 (335 mg) as abrown-yellow solid. This solid was dissolved in CH₂Cl₂ (2.5 ml), and tothis was added TFA (2.5 ml). The mixture was stirred at room temperaturefor 1.5 h and was concentrated to afford 1180-3 (300 mg) as a yellowsolid. FAB-MS=278.

C. To a solution of 1180-3 (28 mg, 0.1 mmol) in DMF (0.25 ml) was addedEDC (60 mg, 0.31 mmol) and DMAP (55 mg). The mixture was stirred at roomtemperature for 10 min. and to this was added amine-TFA salt β-3 (23 mg,0.051 mmol). The resulting reaction mixture was stirred at roomtemperature for 16 h. The usual workup (5% citric acid, 5% NaHCO₃, sat.NaCl) drying (Na₂SO₄), and concentration gave crude 1180-4 (22 mg, 72%).FAB-MS=596.

D. The crude 1180-4 was hydrolyzed as described in Example 1B to givecrude Bio-1180. Purification of the crude product by HPLC afforded pureBIO-1180. HPLC: Room temperature; 26.3 min. >99% purity. FAB-MS=582. ¹HNMR: (DMSO-D⁶, 300 MHz, ppm) 9.00 (br, s, 1H), 8.52 (d, 2H, J=8.3 Hz),8.24 (d, 2H, J=8.3 Hz), 7.50-7.47 (m, 3H), 7.28 (d, 2H, J=8.5 Hz), 7.20(1H, d, J=3.5 Hz), 6.95-6.81 (m, 3H), 6.08 (d, 1H, J=1.4), 5.19-5.16 (m,1H), 4.4-4.2 (m, 1H), 3.51 (dd, J=14.1 Hz and 23.8 Hz), 2.76-2.65 (m,2H), 1.57-1.50 (m, 1H), 1.50-1.44 (m, 2H), 0.92 (d, 2H, J=6.3 Hz), 0.86(d, J=6.3 Hz).

EXAMPLE 47 Synthesis of BIO-1199

To a solution of BIO-1089 (15 mg) in DMSO (1.0 ml), H₂O (2 ml) was addedOxone® (20 mg) and the mixture was stirred at room temperature. The HPLCtrace showed that Bio-1089 (Room temperature=20 min) was disappearingand a new peak (retention time=16.9 min) was forming. After stirring atroom temperature for 16 h, the starting Bio-1089 was almost totallyconsumed. Bio-1199 (Room temperature=16.9 min) was isolated by HPLC andwas >99% pure. FAB-MS=595.

EXAMPLE 48 Synthesis of BIO-1207

A. Procedure C was carried out using amine β-5 (220 mg, 1.053 mmol),this product was then subjected to the conditions descibed in ProcedureD1 to afford 1207-1 (383 mg, 88% for two steps).

B. The method of Example 1A was followed using p-Cbz-aminophenylaceticacid (260 mg, 0.91 mmol) and amine 1207-1 (375 mg, 0.86 mmol) (treatedwith Et₃N) afford 1207-2 (415 mg, 82%) as a pale brown solid.

C. Compound 1207-2 (390 mg, 0.66 mmol) was deprotected as described inProcedure D2 to afford 1207-3 (140 mg, 47%) as a pale brown solid.

D. To a solution of 2-isopropylaniline (135 mg, 1.0 mmol) in CH₂Cl₂ (2ml) and Et₃N (0.5 ml) was added COCl₂ (1.6 ml of 1.9 M solution intoluene, 3.0 mmol) solution at 0° C. slowly and the resulting mixturewas stirred at room temperature for 1 h and diluted with ether (15 ml).Removal of the solid thus formed and the solvents gave 1207-4 (165 mg)as a brown liquid. ¹H NMR: (CDCl₃, 300 MHz, ppm) 7.87-7.64 (m, 4H),3.83-3.74 (m, 1H), 1.81 (d, 6H).

E. To a solution of 1207-4 (12 mg, 0.074 mmol) in DMF (0.12 ml) wasadded 1 drop of Et₃N and 1207-3 (28 mg, 0.062 mmol). The resultingmixture was stirred for 1 h (FAB-MS=617) and was added to methanol (2ml) and 2M LiOH (0.25 ml). This mixture was stirred at room temperaturefor 16 h and was subjected to HPLC. The pure fractions were collectedand concentrated to afford BIO-1207 as a white solid. FAB-MS=603. HPLC:Room temperature=31.2 min; >98.5% purity.

EXAMPLE 49 Synthesis of BIO-1210

The procedure described in Example 22D was followed using2-methylphenylureaphenylacetic acid and the free amine of the TFA-aminesalt prepared in Example 44B (65 mg). The resulting product wassubjected to HPLC. The pure fractions were collected and concentrated toafford BIO-1210 as a white solid. FAB-MS=603. HPLC: Roomtemperature=28.6 min, >99% purity.

EXAMPLE 50 Synthesis of BIO-1224

A. Procedure C was performed using amine β-4 (48 mg, 0.2 mmol) to afford1224-1 (82 mg, 91%) as a white solid. ¹H NMR: (CDCl₃, 300 MHz, ppm)7.49-7.39 (1H), 6.73-6.62 (m, 3H), 5.35-5.28 (m, 1H), 5.19-5.06 (m, 1H),4.16-4.08 (m, 1H), 3.74 (s, 3H), 3.69 (s, 3H), 2.72-2.51 (m, 2H),2.40-2.36 (m, 2H), 1.98-1.75 (m, 2H), 1.90 (s, 3H), 1.28 (s, 9H), 1.19(s, 9H).

B. Compound 1224-1 (60 mg, 0.13 mmol) was dissolved in CH₂Cl₂ (1.5 ml)and TFA (1.5 ml). The mixture was stirred room temperature for 5 h andthe solvents were removed to give 1224-2 as a TFA salt. This compoundwas used in the next step without purification.

¹H NMR: (CDCl₃, 300 MHz, ppm) 7.92 (br, 1H), 6.82-6.78 (m, 3H),5.44-5.26 (m, 1H), 4.40-4.28 (m, 1H), 3.84-3.72 (m, 6H), 2.92-2.70 (m,4H), 2.60-2.25 (m, 2H), 1.92 (s, 3H).

C. The method described in Example 22D was followed using2-methylphenylureaphenylacetic acid (37 mg, 0.13 mmol) and amine 1224-2(60 mg, 0.13 mmol). The resulting product was subjected to HPLC. Thepure fractions were collected and dried to afford BIO-1224 (22 mg, 22%)as a white solid. FAD-MS=623. HPLC: Room temperature=23.8 min, >99%purity. ¹H NMR: (CDCl₃, 300 MHz, ppm) 7.38 (d, 1H), 6.98 (d, 2H), 6.74(d, 2H), 6.72 (m, 2H), 6.51 (t, 1H), 6.43-6.40 (m, 1H), 6.35-6.31 (m,1H), 4.84-4.76 (m, 1H), 4.04-3.97 (m, 1H), 3.39 (s, 6H), 3.33 (s, 2H),2.36-2.18 (m, 2H), 1.91-1.75 (m, 2H), 1.72 (s, 3H), 1.19-0.99 (m, 2H),0.46-0.37 (m, 6H).

EXAMPLE 51 Synthesis of Compound BIO-1056

A. A mixture of 3-methoxy-4-nitrobenzoic acid (2.01 g, 10.2 mmol) andthionyl chloride (2.3 mL, 31.5 mmol) was stirred at 80-90° C. for 1.5 h.The reaction was concentrated and the residue diluted with ether. Theorganic solution was washed with sat. aq. NaHCO₃ (2×), H₂O , then sat.aq. NaCl, dried (MgSO₄) and concentrated to afford3-methoxy-4-nitrobenzoyl chloride (1.92 g, 87%) as a white solid: ¹H NMR(CDCl₃, 300 MHz, ppm) 7.95-7.70 (m, 3H), 4.06 (s, 3H).

B. To a cold (0° C.) solution of TMSCHN₂ (2 M in hexane, 1.5 mL, 3.0mmol) and triethylamine (420 μL, 3.0 mmol) was added a solution of3-methoxy-4-nitrobenzoyl chloride (0.52 g, 2.4 mmol) in acetonitrile(8.5 mL). The reaction was stirred at 0° C. for 24 h and thenconcentrated. The residue was slurried with sat. aq. NaHCO₃ and themixture extracted with ether (3×). The combined ether washes were washedwith water, then sat. aq. NaCl, dried (MgSO₄) and concentrated to afford

ω-diazo-3-methoxy-4-nitroacetophenone (0.53 g, 100%) as a yellow foam:¹H NMR (CDCl₃, 300 MHz, ppm) 7.88 (d, 10 Hz, 1H), 7.61 (s, 1H), 7.27 (d,10 Hz, 1H), 5.97 (s, 1H), 4.02 (s, 3H).

C. To a refluxing solution of ω-diazo-3-methoxy-4-nitroacetophenone(7.95 g, 35.9 mmol) in tBuOH (100 mL) was added a filtered solution ofsilver benzoate (2.50 g, 10.9 mmol) in triethylamine (15 mL) dropwiseover 1 h. After refluxing for 45 min, decolorizing carbon was added andthe hot mixture filtered thru a pad of Celite. The filtrate wasconcentrated and the residue diluted with ethyl acetate. The organicsolution was washed with 5% aq. NaHCO₃ (2×), H₂O, 5% aq. citric acid,H₂O , then sat. aq. NaCl, dried (MgSO₄) and concentrated to affordt-butyl 3-methoxy-4-nitrophenylacetate (8.92 g, 93%) as a brown oil: ¹HNMR (CDCl₃, 300 MHz, ppm) 7.83 (d, 8.3 Hz, 1H), 7.03 (s, 1H), 6.93 (d,8.3 Hz, 1H), 3.97 (s, 3H), 3.58 (s, 2H), 1.45 (s, 9H).

D. A mixture of t-butyl 3-methoxy-4-nitrophenylacetate (0.144 g, 0.539mmol) and 10% Pd on carbon (0.155 g) in ethyl acetate (8 mL) andmethanol (2 mL) was stirred under H₂ (40-60 psi) for 2 h. The mixturewas filtered through Celite and the filtrate concentrated to affordt-butyl 4-amino-3-methoxyphenylacetate (0.123 g, 96%) as a light yellowoil: ¹H NMR (CDCl₃, 300 MHz, ppm) 6.70 (m, 3H), 4.04 (bs, 2H), 3.84 (s,3H), 3.42 (s, 2H), 1.43 (s, 9H).

E. To a solution of t-Butyl 4-amino-3-methoxyphenylacetate (0.123 g,0.52 mmol) in methylene chloride (2.0 mL) was added phenyl isocyanate(60 μL, 0.55 mmol). The reaction was stirred for 45 min thenconcentrated to afford t-butyl 3-methoxy-4-phenylureidophenylacetate(0.190 g, 100%) as a pale yellow foam: ¹H NMR (CDCl₃, 300 MHz, ppm) 8.00(d, 11 Hz, 1H) 7.65-6.94 (m, 7H), 6.80 (d, 9.0 Hz, 1H), 6.74 (s, 1H),3.68 (s, 3H), 3.45 (s, 2H), 1.44 (s, 9H).

F. A solution of t-butyl 3-methoxy-4-phenylureidophenylacetate (0.108 g,0.303 mmol) in trifluoroacetic acid (5.0 mL) was stirred for 30 min. Thereaction was concentrated and the residue coevaporated with methylenechloride (2×) then ether to afford 3-methoxy-4-phenylureidophenylaceticacid (0.090 g, 99%) as a white foam: ¹H NMR (CD₃SOCD₃, 300 MHz, ppm)9.28 (s,1H), 8.18 (s, 1H), 8.02 (d, 7.5 Hz, 1H), 7.58-7.15 (m, 5H), 6.91(bm, 2H), 6.77 (d, 7.5 Hz, 1H), 3.85 (s, 3H), 3.49 (s, 2H).

G. A solution of 3-methoxy-4-phenylureidophenylacetic acid (0.33 g, 0.88mmol), Leu-β-2, prepared utilizing procedures C and D, (0.27 g, 0.90mmol), BOP 0.39 g, 0.90 mmol) and DIPEA (0.77 mL, 4.4 mmol) in DMF (5mL) was stirred for 18 h. The reaction was diluted with ethyl acetateand washed with 60% sat. aq. NaHCO₃ (3×), H₂O, 5% aq. citric acid (3×),H₂O , then sat. aq. NaCl, dried (MgSO₄) and concentrated to afford crudeproduct (0.49 g). The crude product was purified by flash chromatography(silica gel, 1:4 hexane-ethyl acetate) to give BIO-1056 t-butyl ester(0.35 g, 60%) as a white foam: ¹H NMR (CDCl₃, 300 MHz, ppm) 8.00 (d,8.1Hz, 1H), 7.55-7.20 (m, 8H), 7.05 (m, 1H), 6.70 (m, 5H), 5.89 (s, 2H),5.18 (m, 1H), 4.50 (s, 1H), 3.63 (s, 3H), 3.47 (s, 2H), 2.67 (m, 2H),1.68-1.40 (bm, 3H), 1.33 (s, 9H).

H. To a cold (0° C.) solution of BIO-1056 t-butyl ester (0.35 g, 0.53mmol) in methylene chloride (5.0 mL) was added trifluoroacetic acid (5.0mL). The reaction was allowed to warm to RT and stirred for 1 h thenconcentrated to afford crude BIO-1056 (0.315 g). The crude product waspurified by HPLC in two portions to give BIO-1056 (0.16 g, 50%) as awhite solid: ¹H NMR (CD₃SOCD₃, 300 MHz, ppm) 9.25 (s, 1H), 8.43 (d, 8.2Hz, 1H), 8.15 (m, 2H), 8.01 (d, 8.2 Hz, 1H), 7.50-6.55 (m, 10H), 5.97(s, 2H), 5.08 (m, 1H), 4.31 (m, 1H), 3.85 (s, 3H), 3.41 (m, 2H), 2.64(m, 2H), 1.55-1.22 (bm, 3H), 0.80 (m, 6H); HPLC (Gradient A), 35.2 min,(Gradient B), 19.4 min; MS, m/z 605.

EXAMPLE 52 Synthesis of Compound BIO-1221

A. To a solution of t-butyl 4-amino-3-methoxyphenylacetate (0.024 g,0.10 mmol) in methylene chloride (2.0 mL) was added o-tolyl isocyanate(15 μL, 0.12 mmol). The reaction was stirred for 2 h then concentratedto afford t-Butyl 3-methoxy-4-o-tolylureidophenylacetate (0.036 g, 97%)as a tan foam:

¹H NMR (CDCl₃, 300 MHz, ppm) 8.05 (d, 7.9 Hz, 1H), 7.55 (d, 7.9 Hz, 1H),7.45-7.05 (m, 5H), 6.78 (m, 2H), 3.73 (s, 3H), 3.48 (s, 2H), 2.23 (s,3H), 1.44 (s, 9H).

B. A solution of t-butyl 3-methoxy-4-o-tolylureidophenylacetate (0.016g, 0.043 mmol) in trifluoroacetic acid (1.0 mL) was stirred for 1 h. Thereaction was concentrated and the residue coevaporated with methylenechloride (2×) then ether to afford 3-methoxy-4-o-tolylureidophenylaceticacid (0.0135 g, 100%) as a white residue.

C. The procedure described in Example 51G was performed using3-methoxy-4-o-tolylureidophenylacetic acid (0.0135 g, 0.043 mmol) andamine salt prepared from β-3 utilizing procedures C and D (0.0185 g,0.041 mmol)to afford BIO1221 methyl ester (0.016 g, 60%) as a whitefoam: ¹H NMR (CDCl₃, 300 MHz, ppm) 8.10 (d, 1H), 7.61 (d, 1H), 7.45-7.00(m, 7H), 6.85-6.65 (m, 5H), 5.93 (s, 2H), 5.20 (m, 1H), 4.37 (m, 1H),3.85 (s, 3H), 3.61 (s, 3H), 3.52 (s, 2H), 2.75 (m, 2H), 2.30 (s,3H),1.65-1.10 (bm, 3H), 0.86 (m, 6H).

D. BIO1221 methyl ester (0.016 g, 0.025 mmol) was hydrolyzed using themethod described in Example 1B to give BIO-1221 (0.0087 g, 56%) as awhite powder: ¹H NMR (CDCl₃, 300 MHz, ppm) 7.93 (d, 1H), 7.70 (d, 1H),7.49 (d, 1H), 7.37-6.92 (m, 6H), 6.78-6.55 (m, 5H) 5.81 (s, 2H), 5.09(m, 1H), 4.27 (m, 1H), 3.73 (s, 3H), 3.40 (s, 2H), 2.58 (m, 2H), 2.19(s, 3H), 1.48-1.25 (bm, 3H), 0.76 (m, 6H); HPLC (Gradient A), 35.2 min;MS, m/z 619.

EXAMPLE 53 Synthesis of Compound BIO-1238

A. The procedure described in Example 43A was performed using amine β-5to give 1238-1. Yield:92%.

¹HNMR (CDCl₃, 300 MHz, ppm): 7.19 (d, 2H, J=8.6 Hz), 6.82 (d, 2H, J=8.6Hz), 5.36-5.28 (m, 2H), 4.25-4.22 (m, 1H), 3.72 (s, 3H), 3.56 (s, 3H),2.72-2.66 (m, 2H), 2.49-2.41 (m, 2H), 2.1 (s, 3H), 1.92-1.78 (m, 1H),1.48 (s, 9H). The Boc group was removed by TFA/CH₂Cl₂ to afford TFA salt1238-1. 1HNMR (CDCl₃, 300 MHz, ppm): 7.12 (d, 2H, J=8.5 Hz), 6.74 (d,2H, J=8.5 Hz), 5.32 (m, 1H), 4.38 (m, 1H), 3.68 (s, 3 H), 3.51 (s, 3H),2.77-2.69 (m, 2H), 2.55-2.38 (m, 1 H), 2.36-2.31 (m, 1H), 2.16-2.02 (m,2H), 1.91 (s, 3 H).

B. The procedure described in Example 1A was performed using2-methylphenylureaphenylacetic acid (20 mg, 0.7 mmol) and TFA salt1238-1 (30 mg, 0.7 mmol) to give 1238-2 (35 mg, 83%) as a white solid.¹HNMR (DMSO-d⁶, 300 MHz, ppm): 7.91 (d, 1H), 7.52 (d, 2H, J=8.5 Hz),7.35-7.30 (m, 4H), 7.02 (d, 1H), 6.80 (d, 2H, J=8.5 Hz), 5.79-5.68 (m,1H), 4.40-4.28 (m, 1 H), 3.71 (s, 3H), 3.63 (s, 3H), 3.35-3.38 (m, 2H),2.49 (br, s, 2H), 2.00 (s 3H).

C. A solution of 1238-2 (20 mg, 0.033 mmol) in MeOH (3 mL) and aqueousLiOH (3 mL of 2N) was stirred at room temperature overnight, thereaction mixture was cooled to 0° C. and acidified by adding TFA untilpH=3-4 (pH paper). The desired product was isolated and purified by LC(Vydac C18 column; gradient 8) to give 12 mg (0.017 mmol; 61%) ofBIO-1238 as a white solid: FAB-MS=595.

EXAMPLE 54 Synthesis of Compound BIO-1245

A. 1245-1 was prepared from commercially available N-BOC-methioninesulfone (562 mg, 2.0 mmol) and amine β-3 (470 mg, 2.10 mmol) using themethod described in Example 1A to afford crude 1245-1 (962 mg, 1.90mmol, 95%) as a white foam which was used without further purification.¹HNMR(CDCl₃): δ 7.31 (1H, d, J=8.3 Hz), 6.77-6.7(3H, m), 5.91(2H, s),5.04(1H, d, J=7.6 Hz), 5.27(1H, m), 4.30(1H, br), 3.61(3H, s), 3.15(1H,m), 2.93(1H, m), 2.89(3H, s), 2,85(2H, m), 2.22(2H, m), 1.42(9H, s).

B. Compound 1245-1 (962 mg, 1.90 mmol) was treated with 4N HCl/dioxaneas the reagent. Concentration affords the hydrochloride salt 1245-2 as awhite solid (800 mg, 1.89 mg, 1.89 mmol, 99%) which was used withoutfurther purification. ¹HNMR(CDCl₃): δ 8.75(1H,br), 8.20(2H, br),6.91-6.55(3H,m), 5.90(2H, bs), 5.42(1H, br), 4.55(1H, br) 3.60(3H, s),3.45-3.0(2H, bm), 2.90 (3H, s), 2.85-2.40(4H, bm).

C. The procedure described in Example 22D was performed using compound1245-2 (800 mg, 1.89 mmol) and o-methylphenylureaphenyl acetic acid (543mg, 1.89 mmol) to afford crude 1245-3 (1.15 g, 1.76 mmol, 93%) as awhite solid which used without further purification.

¹HNMR(DMSO=d₆): δ 7.95(1H,s), 7.89(1H,d,J=7.9 Hz), 7.43(2H, D, J=7.9Hz), 7.20(4H, m), 7.00-6.78(4H, m), 6.03(2H,s), 5.18(1H, m), 4.40(1H,m), 3.58(3H, s), 3.49(3H, s), 3.39(2H, br), 2.90-2.49 (2H, m), 2.29(3H,s), 2.00(2H, m).

D. Compound 1245-3 (1.1 g, 1.7 mmol) was hydrolyzed as described inExample 1B to afford crude BIO-1245 (490 mg, 0.77 mmol, 45%) as a whitesolid >90% pure by HPLC. A small amount (^(˜)150 mg) was purified byprep HPLC to afford pure BIO-1245(81 mg, 54% recovery) as a white solidm/z=639(100% pure by HPLC).

¹HNMR(DMSO-d₆): δ 8.60(0.5H, bs), 8.57(1H, d, J=8.lHz), 8.37(1H, d,J=8.lHz), 8.18(1H, s), 8.05(0.5H, s), 7.89(1H, d, J=8.0 Hz), 7.43(2H, d,J=8.04z), 7.21(4H, m), 6.97-6.81(4H, m), 6.03(2H, s), 5.13(1H,m),4.43(1H, m), 3.80(1H, br), 3.49(3H, s), 2.93(2H, m), 2.45(2H, m),2.30(3H, s), 2.01(2H, m).

EXAMPLE 55 Synthesis of BIO-1246

A. To a suspension of L-cysteine (1.5 g, 12.4 mmol) in methanol (8 mL)was added excess sodium methoxide (2.0 g, 37.2 mmol) followed by acatalytic amount of sodium iodide (^(˜)100 mg). After stirring at roomtemperature for 30 min. 1-bromo-2-propanol (1.7 g, 12.4 mmol) was addedand the reaction was stirred overnight. The reaction mixture was thenneutralized to pH^(˜)7, diluted with water (20 mL) and concentrated toremove the methanol. The solution was then diluted with dioxane (20 mL)and triethylamine (7.0 mL, 50 mmol) was added followed by BOCON (3.1 g,12.4 mmol) and the reaction was stirred at room temperature for 3 h. Thereaction was worked up by diluting with water (20 mL) and extractingwith ethyl acetate (3×25 mL). The organic extracts were discarded andthe aqueous solution acidified to pH=1 with 1N HCl. The aqueous wasextracted with ethyl acetate (4×30 mL), dried over sodium sulfate andconcentrated to afford 1246-1 (2.87 g, 10.4 mmol, 83%, 2 steps) as athick pale yellow syrup. ¹HNMR(CDCl₃) δ 5.60-5.50(1H, br), 4.60-4.50(1H,br), 4.44(2H, t, J=6.3 Hz), 3.02(2H, bm), 32.65(2H,br) 2.03 (2H,M),1.45(9H, S).

B. The procedure of Example 1A was performed using 1246-1 (33 mg, 0.11mmol) and amine β-3 (22 mg, 0.10 mmol) to afford 1246-2 (39 mg, 0.08mmol, 80%) as a pale yellow foam which was used without purification inthe next step. ¹HNMR(CDCl₃): δ 6.80-6.60(3H,m), 5.91(2H,s), 5.50(1H,bm),4.35(1H,bm), 3.71, (2H,bt), 3.61(3H, s), 3.15-2.65(6H,m), 1.85(2H,m),1.46(9H, s).

C. Compound 1246-2 (39 mg, 0.08 mmol) was treated with TFA to give thecorresponding amine-TFA salt of 1246-2 which was subjected to theconditions described in Example 54C to give a white solid which wasdirectly hydrolysed as described in Example 1B to the free acid. A smallaliquot was purified by HPLC. The clean fractions were collected toafford BIO-1246(^(˜)3 mg) M/Z=637(100% pure by HPLC) as a white solid.

¹HNMR(DMSO-d₆): δ 9.01(1H,s), 8.66(1H,d,J=5.3 Hz), 8.30(1H,d,J=5.5 Hz),7.94(1H,s), 7.88(1H,d,J=5.3 Hz), 7.42(2H,d,J=5.5 Hz), 7.20-7.15(4H,m),7.00-6.94(2H,m), 6.88-6.79(2H,m), 6.02(2H,s), 5.12(1H,m), 4.48(1H,m),3.65(2H,m), 2.90-2.45(6H,m), 2.28(3H,s), 1.65(2H,m).

EXAMPLE 56 Synthesis of BIO-1248

A. A mixture of 4-fluorobenzaldehyde (2.48 g; 20 mmol), malonic acid(2.5 g, 24 mmol) and ammonium acetate (2.16 g; 28 mmol) in ethanol (100mL) was refluxed under argon overnight. After cooling to roomtemperature, the solid precipitate was collected by filtration andwashed with ethanol (3×30 mL) and dried under vacuum to give 1.0 g (27%)of white solid, which was used without further purification.

To a suspension of the white solid (1.0 g, 9.4 mmol) in methanol wasadded SOCl₂ (6.01 mmol; 5.2 mL of 2 M in CH₂Cl₂). The resultant solutionwas stirred at room temperature overnight. After removal of excesssolvent, the residue was dissolved in EtOAc, basified with sat. NaHCO₃,and dried with Na₂SO₄. The organic solution was concentrated underreduced pressure to give 900 mg (84%) of amine 1248-1 as a light yellowoil: ¹H NMR (CDCl₃, 300 MHz, ppm) 7.28 (m, 2 H, Ar), 6.96 (m, 2 H, Ar),4.46 (t, J=6.8, 1 H), 3.62 (s, 3 H, OMe), 2.58 (d, J=6.8 Hz, 2 H), 1.69(s, 2 H, NH); TLC, 10% MeOH/CH₂Cl₂, R_(f)=0.5.

B. Amine 1248-1 (300 mg, 1.52 mmol) was coupled withNα-t-Boc-Nε-leu-N-hydroxysuccinimide (300 mg, 1.52 mmol) using themethod described in Procedure C. The resulting adduct was deprotectedwith trifluoroacetic acid and, then basified with Et₃N as described inProcedure D1 to give the amine 1248-2 in 84%: ¹H NMR (CDCl₃, 300 MHz,ppm) 8.20 (d, J=7.1 Hz, 1 H), 7.24 (m, 2 H, Ar), 6.97 (m, 2 H, Ar), 5.33(m, 1 H), 3.58 (s, 3 H, OMe), 3.38 (m, 1 H), 2.82 (m, 2 H), 1.66 (m, 2H), 1.30 (m, 1 H), 1.22 (s, 2 H), 0.91 (m, 6 H); TLC, 10% MeOH/CH₂Cl₂,R_(f)=0.47 and 0.38.

C. 2-Methylphenylureaphenylacetic acid (77 mg, 0.27 mmol) was coupledwith amine 1248-2 (70 mg, 0.23 mmol) using the method described inExample 22D to give 1248-3 in 61% yield. ¹H NMR (DMSO-d⁶, 300 MHz, ppm)δ9.15 (d, J=5.9 Hz, 1 H), 8.53 (t, J=7.5 Hz, 1 H) 8.17 (d, J=8.2 Hz,1H), 8.0 (s, 1 H), 7.84 (d, J=8.0 Hz, 2 H), 7.35 (m, 4 H), 7.13 (m, 6H), 6.92 (t, J=8.2 Hz, 1 H), 5.20 (m, 1 H), 4.30 (m, 1 H), 3.52 (s, twopeaks, 3 H, OMe), 3.45-3.24 (m, 2 H), 2.75 (m, 2 H), 2.24(s, 3 H, Me),1.57-1.33 (m, 3 H), 0.82 (m, 6 H); HPLC (gradient 1**) 21.2 min and 21.5min (1:24); FABMS, m/z 577 (C₃₃H₃₇N₄O₅F of M⁺+1 requires 577).

D. A solution of 1248-3 (22 mg, 0.038 mmol) in DMSO (1 mL) and MeOH (2mL) was hydrolyzed with aqueous LiOH as described in Example 1B. Theproduct was purified on a Vydac reverse-phase C18 column (22 mm×25 cm)using a linear gradient of 15% CH₃CN/H₂O (0.1% TFA) to 40% CH₃CN/H₂O(0.1% TFA) with a flow rate of 10 mL/min to give BIO-1248 in 29%isolated yield. ¹H NMR (DMSO-d⁶, 300 MHz, ppm) δ 8.93 (s,1 H), 8.46 (d,J=8.3 Hz, 1 H), 8.25 (d, J=8.2 Hz,1 H), 7.87 (s, 1 H), 7.82 (d, J=8.0Hz, 1 H), 7.33 (m, 5 H), 7.12 (m, 5 H), 6.93 (m, 1 H), 5.15 (m, 1 H),4.28 (m, 1 H), 3.35 (m, 2 H), 2.65 (d, J=7.2 Hz, 2 H), 2.22 (s, 3 H,Me), 1.55 (m, 1 H), 1.43 (m, 2 H), 0.83 (m, 6 H); HPLC (gradient 1) 18.7min and 19.3 min (1:24); FABMS, m/z 563 (C₃₁H₃₅N₄O₅F of M⁺+1 requires563).

EXAMPLE 57 Synthesis of BIO-1270

A. Amine β-3 (500 mg, 2.24 mmol) was coupled withNα-Cbz-Nε-t-Boc-_(L)-Lys-N-hydroxysuccinimide (1.0 g, 2.1 mmol) usingProcedure C to give the coupled adduct 1270-1 (1.1 g, 82%). This adductwas deprotected with trifluoroacetic acid and was basified with Et₃N aspreviously described in Procedure D to give 1270-2 in 54% yield. ¹H NMR(CDCl₃, 300 MHz, ppm) δ 7.31 (m, 6 H), 6.72 (m, 3 H), 5.90 (s, 2 H),5.58 (d, J=9 Hz, 1 H), 5.26 (m, 1 H), 5.07 (s, 2 H), 4.15 (m, 1 H), 3.58(s, 3 H, OMe), 2.77 (m, 2 H), 2.61 (m, 2 H), 1.79 (m, 1 H), 1.59 (m, 1H), 1.41-1.30 (m, 6H); TLC, 10% MeOH/CH₂Cl₂, R_(f)=0.11.

B. To a stirred solution of 1270-2 (15.5 mg, 0.032 mmol) and pyridine(10.1 mg, 0.128 mmol) in CH₂Cl₂ at rt is added acetyl chloride (7.5 mg,0.096 mmol). After stirring for 3 hours the reaction is concentrated andreverse phase chromatography provided 1270-3 (16.3 mg, 95%) as a whitefoam. ¹HNMR(CDCl₃, 300 MHz, ppm) 7.32(S, 5H), 6.70(m, 3H) 5.91(s, 2H),5.82(m, 1H), 5.55(m, 1H), 5.25 (m, 1H), 5.09(s, 1H), 4.13(m, 1H),3.60(S, 3H), 3.28(M, 2H), 2.9-2.4(m, 3H), 1.94(S, 3H), 1.9-1.76(m, 1H)1.70-1.58 (m, 1H),1.52-1.42(m, 2H), 1.36-1.22(m, 2H).

C. Procedure D2 was performed using 1270-3 (reaction progress wasfollowed by HPLC) to give compound 1270-4 (14.1 mg, quantitative yield)as a clear oil which was used as the crude material.

D. The procedure of Example 54C was performed using 1270-4 (14.1 mg,0.036 mmol). Purification was carried out via preperative HPLC andprovided Bio 1270-OMe (9.1 mg, 38%) as a white solid. ¹HNMR(DMSO_(D6),300 MHz, ppm), 8.13(d, 1H J=10.3S), 8.03(s, 1H), 7.93(d, 1H J=10.35),7.83(m, 1H), 7.49(d, 2H J=10.35), 7.28(m, 5H), 7.10-6.81(m. 5H), 6.08(s,2H), 5.20(dd, 1H J=9.66, 17.25), 4.33(dd, 1H J=8.97, 15.18), 3.63(s,3H), 3.5(s, 2H), 3.1-2.95(m, 2H), 2.85-2.74(m, 2H), 2.33(s, 3H), 1.86(s,3H), 1.72-1.49(m, 2H), 1.5-1.32(m, 3H), 1.31-1.09(m, 2H).

E. To Bio 1270-OMe (9.1 mg, 0.016) in 1 ml of DMSO_(D6) (NMR sample) wasadded 20 ul of 2N LiOH(0.041 mmol) and the reaction was stirred at rt.overnight. The reaction was acidified (red to litmus) with 3 drops ofTFA and purified by preparative HPLC This afforded BIO-1270 (6.2 mg,60%) as a white solid.

¹HNMR(DMSO_(D6), 300 MHz, ppm), 8.5(d, 12H J=10.35), 8.19(d, 1HJ=10.35), 7.99(s, 1H), 7.93(d, 1H J=10.35), 7.82 (m, 1H), 7.45(d, 2HJ=10.35). 7.28(m, 4H), 7.05(m, 1H), 6.98-6.89 (m, 2H) 6.86(m, 1H),6.09(S, 2H), 5.66(dd, 1H J=8.28, 16.56) 4.32(dd, 1H J=7.59, 13,8), 3.27(s, 2H), 2.98 (m, 2H) 2.75(m, 2H), 2.33 (s, 3H), 1.87(s, 3H), 1.69-1.48(m, 2H), 1.46-1.32(m, 3H), 1.28-1.12 (m, 2H); MS, m/z 646; HPLC(Gradient 1) 19.73 min. 100%.

Gradient 3 15% B 65% B 50 min.

Gradient 1 20% B 70% B 50 min.

EXAMPLE 58 Synthesis of BIO-1282

A. A solution of ethyl 3-pyridylacetate (1.65 g, 9.90 mmol) in 32%peracetic acid (10 mL) was stirred at 80-90° C. for 2 h. The reactionwas concentrated and the residue coevaporated with methanol (2×) thenmethylene chloride to afford ethyl 3-pyridylacetate N-oxide (1.80 g,100%) as a white solid: ¹H NMR (CDCl₃, 300 MHz, ppm) 8.38 (s, 1H), 8.22(d, 1H), 7.39 (d, 1H), 4.20 (q, 2H), 3.62 (s, 2H), 1.26 (t, 3H).

B. A solution of salicylamide (4.14 g, 30.2 mmol) and conc. sulfuricacid (3 drops) in acetone (40 mL) was refluxed for 5 h. The reaction wasconcentrated and the residue taken up in ethyl acetate. The organicsolution was washed with 1 N NaOH (2×), 1 N HCl (2×), H₂O , then sat.aq. NaCl, dried (MgSO₄) and concentrated to afford2,2-dimethyl-4-keto-1,3-benzoxazine (2.50 g, 47%) as a white solid: ¹HNMR (CDCl₃, 300 MHz, ppm) 7.92 (d, 1H), 7.60 (bs, 1H), 7.47 (m, 1H),7.06 (m, 1H), 6.92 (d, 1H), 1.65 (s, 6H).

C. A solution of 2,2-dimethyl-4-keto-1,3-benzoxazine (1.77 g, 10.0 mmol)and PCl₅ (2.09 g, 10.0 mmol) in POCl₃ (3.0 mL) was stirred at RT for 1 hthen at 50-60° C. for 2 h. The reaction was concentrated and the productdistilled (90-95° C./2-3 mm Hg) to afford4-chloro-2,2-dimethyl-3H-1,3-benzoxazine. (0.496 g, 25%) as a clear oil:¹H NMR (CDCl₃, 300 MHz, ppm) 7.58 (d, 1H), 7.48 (m, 1H), 6.97 (m, 1H),6.94 (d, 1H), 1.63 (s, 6H).

D. A mixture of 4-chloro-2,2-dimethyl-3H-1,3-benzoxazine (0.145 g, 0.741mmol) and ethyl 3-pyridylacetate N-oxide (0.270 g, 1.49 mmol) inmethylene chloride (5.0 mL) was refluxed for 20 h. The reaction wasconcentrated and the residue taken up in ethyl acetate. The organicmixture was washed with 60% sat. aq. NaHCO₃ (2×), H₂O , sat. aq. NaCl,dried (MgSO₄), and concentrated to afford an oily residue (0.148 g).

The crude oily residue (0.148 g) in conc. HCl (10 mL) was refluxed for18 h. The reaction was concentrated and the residue partitioned in H₂Oand methylene chloride. The aqueous solution was washed with methylenechloride (2×) and then concentrated to afford a white solid (0.105 g).

A solution of the white solid (0.105 g) in methanol (5.0 mL) was treatedwith thionyl chloride (0.5 mL, 7 mmol) dropwise over 30 min. Thereaction was stirred for 2 h then concentrated. The residue was taken upin 5% aq. NH₄OH and extracted with methylene chloride (3×). The organicextracts were dried (MgSO₄) and concentrated to afford methyl5-(2-aminopyridyl)acetate (0.012 g, 10% for three steps) as a whitesolid: ¹H NMR (CDCl₃, 300 MHz, ppm) 7.93 (s, 1H), 7.40 (d, 1H), 6.50 (d,1H), 4.52 (bs, 2H), 3.70 (s, 3H), 3.49 (s, 2H); MS, m/z 167.

E. To a solution of methyl 5-(2-aminopyridyl)acetate (0.012 g, 0.072mmol) in methylene chloride (1.0 mL) was added o-tolyl isocyanate (10μL, 0.081 mmol). The reaction was stirred for 1 h then concentrated toafford a white residue (0.020 g) containing methyl5-(2-o-tolylureido)pyridylacetate.

F. A solution of crude methyl 5-(2-o-tolylureido)pyridylacetate (0.020g) in methanol (1.0 mL) was treated with 2 M LiOH (100 μL, 0.20 mmol).The reaction was stirred for 18 h then concentrated. The crude productwas purified by HPLC to afford 5-(2-o-tolylureido)pyridylacetic acid(0.013 g, 65%) as a white powder: ¹H NMR (CDCl₃, 300 MHz, ppm) 8.10 (s,1H), 7.87 (bd, 1H), 7.75 (bd, 1H), 7.21 (mn, 1H), 7.08 (m, 1H), 3.62 (s,2H), 2.38 (s, 3H); MS, m/z 286.

G. The procedure described in Example 1A was performed using5-(2-o-tolylureido)pyridylacetic acid (0.013 g, 0.045 mmol) and theamine prepared in Example 14A (0.022 g, 0.049 mmol) to afford BIO-1282methyl ester (0.020 g, 60%): ¹H NMR (CDCl₃, 300 MHz, ppm) 8.18-7.73 (m,4H), 7.55 (d, 1H), 7.35-6.65 (m, 10H), 5.93 (s, 1H), 5.28 (m, 1H), 4.45(m, 1H), 3.69-3.45 (m, 5H), 2.81 (bm, 2H), 2.20 (s, 3H), 1.54 (bm, 3H),0.92 (m, 6H).

H. To a mixture of BIO1282 methyl ester (0.020 g, 0.033 mmol) inmethanol (02.0 mL) was added 2.0 M LiOH (200 μL, 0.40 mmol). Thereaction was stirred for 20 h then concentrated. The residue (containinga 4:5 mixture of BIO1282 and starting ester) was dissolved in DMF (0.5mL) and methanol (0.5 mL) then stirred for an additional 28 h. Thereaction was acidified with trifluoroacetic acid and concentrated. Thecrude product was purified by HPLC to give BIO-1282 (0.0056 g, 24%) as awhite powder: ¹H NMR (CDCl₃, 300 MHz, ppm) 8.44 (d, 8.1 Hz, 1H), 8.26(d, 8.3 Hz, 1H), 8.15 (s, 1H), 8.04 (d, 8.0 Hz, 1H), 7.66 (d, 8.7 Hz,1H), 7.32-7.13 (m, 3H), 7.05-6.94 (m, 1H), 6.85-6.65 (m, 3H), 5.96 (s,2H), 5.06 (m, 1H), 4.29 (m, 1H), 3.45 (m, 2H) 2.63 (m, 2H), 2.31 (s,3H), 1.57-1.20 (m, 3H), 0.78 (m, 6H); HPLC (Gradient A), 27.0 min; MS,m/z 590.

EXAMPLE 59 Synthesis of BIO-1294

A. To a stirred solution of the amine prepared in Example 57A (102 mg,0.21 mmol) in CH₂Cl₂ (20) was added CH₃SO₂Cl (48 mg, 32 μL, 0.42 mmol)and Et₃N (50 μL). The resulting mixture was stirred at RT for 18 h. Thereaction mixture was diluted with CH₂Cl₂ (40 mL), washed with 5% citricacid (20 mL), H₂O (10 mL), Sat. NaHCO₃ (20 mL), Sat. NaCl (20 mL) anddried with Na₂SO₄. The organic solution was concentrated under reducedpressure to give 110 mg (92%) of 1294-1 as a white solid: ¹H NMR (CDCl₃,300 MHz, ppm) δ 7.30 (m, 6 H), 6.74 (m, 3 H), 5.90 (s, 2 H), 5.70 (m, 1H), 5.25 (m, 1 H), 5.07 (s, 3 H), 4.16 (m, 1 H), 3.58 (s, 3 H, OMe),3.02 (m, 2 H), 2.88 (s, 3 H), 2.75 (m, 2 H), 1.76 (m, 1 H), 1.60 (m, 1H), 1.50 (m, 2 H), 1.32 (m, 2 H); TLC, 10% MeOH/CH₂Cl₂, R_(f)=0.67.

B. To a solution of compound 1294-1 (110 mg,0.195 mmol) was dissolved inmethanol (10 ml) was added acetic acid (0.2 ml) and Pd (OH)₂ (110 mg).The resulting mixture was hydrogenated (H₂, 50 psi) at RT for 48 h.After standard work-up, 1294-2 (35 mg, 42%) was obtained as colorlessoil: ¹H NMR (CDCl₃, 300 MHz, ppm) δ 8.06 (m, 1 H), 6.75 (m, 3 H), 5.92(s, 2 H), 5.25 (m, 1 H), 5.02 (m, 1 H), 3.61 (s, 3 H), 3.35 (m, 1 H),3.10 (m, 2 H), 2.94 (s, 3 H), 2.80 (m, 2 H), 1.87-1.30 (m, 8 H); HPLC(gradient 8) 12 min.

C. 2-Methylphenylureaphenylacetic acid (35 mg, 0.12 mmol) was coupledwith the amine 1294-2 (35 mg, 0.08 mmol) as described in Example 1A togive compound 1294-3 in 88%. ¹H NMR (CDCl₃, 300 MHz, ppm) δ 8.50 (m,1H), 8.30 (s, 1 H), 8.16 (m,1 H), 7.82 (m, 1 H), 7.40 (m, 2 H), 7.22-7.05(m, 5 H), 7.00-6.70 (m, 5 H), 5.98 (s, 2 H), 5.11 (m, 1 H), 4.22 (m, 1H), 3.52 (s, 3 H), 3.36 (m, 2 H), 2.91-2.62 (m, 7 H), 2.25 (s, 3 H),1.60-1.05 (m, 6 H); HPLC (gradient 8) 31 min; FABMS, m/z 696(C₃₄H₄₁N₅O₉S of M⁺+1 requires 696).

D. A solution of compound 1294-3 (50 mg, 0.07 mmol) in MeOH (3 mL) washydrolyzed with aqueous LiOH as previously described. The product waspurified on a Vydac reverse-phase C18 column (22 mm×25 cm) using alinear gradient of 15% CH₃CN/H₂O (0.1% TFA) to 40% CH₃CN/H₂O (0.1% TFA)with a flow rate of 10 mL/min to give BIO-1294 in 41% isolated yield. ¹HNMR (CDCl₃, 300 MHz, ppm) δ 8.95 (m,1 H), 8.42 (d, J=8.2 Hz, 1 H), 8.08(d, J=8.1 Hz, 1 H), 7.88 (s, 1 H), 7.83 (d, J=8.0 Hz, 2 H), 7.36 (d,J=8.2 Hz, 2 H), 7.15 (m, 4 H), 7.10-6.71 (m, 5 H), 5.97 (s, 2 H), 5.04(m, 1 H), 4.22 (m, 1 H), 3.41-3.25 (m, 2 H), 2.83-2.80 (m, 6 H), 2.23(s, 3 H), 1.70-1.04 (m, 6 H); HPLC (gradient 8) 27 min; FABMS, m/z 682(C₃₃H₃₉N₅O₉S of M⁺+1 requires 682).

EXAMPLE 60 Synthesis of BIO-1321

A. A mixture of methyl 4-formylbenzoate (3.48 g; 20 mmol), malonic acid(2.5 g, 24 mmol) and ammonium acetate (2.16 g; 28 mmol) in ethanol (100mL) was refluxed under argon overnight. After cooling to roomtemperature, the solid precipitate was collected by filtration andwashed with ethanol (3×30 mL). The white solid was dried under vacuumovernight to give 2.8 g (63%) of 1321-1.

B. To a suspension of compound 1321-1 (1.0 g, 4.48 mmol) in methanol (50mL) was added SOCl₂ (5.4 mmol; 2.7 mL of 2 M in CH₂Cl₂). The resultantsolution was stirred at room temperature overnight. After removal ofexcess solvent, the residue was dissolved in EtOAc, basified with sat.NaHCO₃, and dried with Na₂SO₄. The organic solution was concentratedunder reduced pressure to give 780 mg (53%) of the amine 1321-2 as alight yellow oil: ¹H NMR (CDCl₃, 300 MHz, ppm) 7.99 (m, 2 H, Ar), 7.56(d, J=8.1 Hz, 1 H, Ar), 7.42 (d, J=8.0 Hz, 1 H, Ar), 4.46 (t, J=6.7, 1H), 3.85 (s, 3 H, OMe), 3.65 (s, 3 H, OMe), 2.65 (d, J=6.8 Hz, 2 H),1.88 (s, 2 H, NH).

C. The amine 1321-2 (500 mg, 1.11 mmol) was coupled withNα-t-Boc-Nε-Leucine-N-Hydroxysuccinimide (380 mg, 1.0 mmol) as describedin Procedure C to give material which was deprotected withtrifluoroacetic acid and, then basified with Et₃N as described inProcedure D1 to give amine 1321-3 in 70% yield: ¹H NMR (CDCl₃, 300 MHz,ppm) 8.32 (t, J=9.1 Hz, 1 H), 8.20 (d, J=8.3 Hz, 2 H), 7.34 (m, 2 H,Ar), 5.40 (m, 1 H), 3.86 (s, 3 H, OMe), 3.58 (s, 3 H, OMe), 3.41 (m, 1H), 2.85 (m, 2 H), 1.67 (m, 2 H), 1.53 (s, 2 H), 1.30 (m, 1 H), 0.90 (m,6 H).

D. 2-Methylphenylureaphenylacetic acid (54 mg, 0.19 mmol) was coupledwith amine 1321-3 (70 mg, 0.23 mmol) using the method described inExample 22D to give the 1321-4 in 87% yield: ¹H NMR (DMSO-d⁶, 300 MHz,ppm) δ8.62 (m, 1 H), 8.18 (d, J=8.1 Hz, 1 H), 8.10 (m, 1H), 7.94-7.82(m, 4 H), 7.48-7.34 (m, 4 H), 7.17-7.13 (m, 4 H), 6.91 (t, J=7.3 Hz, 1H), 5.24 (m, 1 H), 4.30 (m, 1 H), 3.53 (s, two peaks, 3 H, OMe),3.39-3.34 (m, 2 H), 3.05 (m, 2 H), 2.24(s, 3 H, Me), 1.60-1.36 (m, 3 H),0.83 (m, 6 H); HPLC (gradient 8) 40 min (1:1); FABMS, m/z 617(C₃₃H₄₀N₄O₇ of M⁺+1 requires 617).

E. A solution of 1321-4 (70 mg, 0.11 mmol) in DMSO (1 mL) and MeOH (2mL) was hydrolyzed with aqueous LiOH as described in Example 1B. Theproduct was purified on a Vydac reverse-phase C18 column (22 mm×25 cm)using a linear gradient of 15% CH₃CN/H₂O (0.1% TFA) to 40% CH₃CN/H₂O(0.1% TFA) with a flow rate of 10 mL/min to give BIO-1321 (22 mg, 34%isolated yield): ¹H NMR (DMSO-d⁶, 300 MHz, ppm) δ 8.95 (d, J=4.6 Hz, 1H), 8.57 (m, 1 H), 8.13 (d, J=8.3 Hz, 1 H), 7.88-7.81 (m, 4 H),7.44-7.32 (m, 4 H), 7.17-7.10 (m, 4 H), 6.92 (t, J=7.4 Hz, 1 H), 5.20(m, 1 H), 4.31 (m, 1 H), 3.46-3.27 (m, 2 H), 2.70 (m, 2 H), 2.22(s, 3 H,Me), 1.59-1.32 (m, 3 H), 0.81 (m, 6 H); HPLC (gradient 8) 27.8 min and28.1 min (1:1); FABMS, m/z 589 (C₃₁H₃₆N₄O₇ of M⁺+1 requires 589)

EXAMPLE 61 Synthesis of Compound 1336

A. A slurry of 2,6-dichloro-3-nitropyridine (92%, 9.9 g, 47 mmol) and K₂CO₃ powder (6.5 g, 47 mmol) in methanol (100 mL) was stirred for a weekat RT. The reaction was filtered and concentrated. The residue waspartitioned in ethyl acetate and 60% sat. aq. NaHCO₃. The organicsolution was washed with 60% sat. aq. NaHCO₃ (2×), H₂O , then sat. aq.NaCl, dried (MgSO₄) and concentrated to afford2-chloro-6-methoxy-5-nitropyridine and2-chloro-6-methoxy-3-nitropyridine (8.9 g, 100%) as a light yellowsolid: ¹H NMR (CDCl₃, 300 MHz, ppm) 8.31 (d, 8.3 Hz, 1H), 8.28 (d, 8.9Hz, 1H), 7.10 (d, 8.3 Hz, 1H), 6.82 (d, 8.9 Hz, 1H), 4.15 (s, 3H), 4.06(s, 3H).

B. A mixture of 2-chloro-6-methoxy-5-nitropyridine and2-chloro-6-methoxy-3-nitropyridine (8.9 g, 47 mmol), t-butyl methylmalonate (10 mL, 60 mmol), and NaH (95%, 3.1 g, 120 mmol) in THF (250mL) was stirred at RT for 24 h. The reaction was concentrated and theresidue treated with trifluoroacetic acid (200 mL) for 2 h. The reactionwas concentrated and the product separated by flash chromatography(silica gel, 95:5 hexane-ethyl acetate) to afford methyl6-(2-methoxy-3-nitro)pyridylacetate (3.3 g, 62%) as a yellow oil: ¹H NMR(CDCl₃, 300 MHz, ppm) 8.27 (d, 8.0 Hz, 1H), 7.04 (d, 8.0 Hz, 1H), 4.09(s, 3H), 3.85 (s, 2H), 3.75 (s, 3H).

C. A mixture of methyl 6-(2-methoxy-3-nitro)pyridylacetate (0.047 g,0.21 mmol) and 10% Pd on carbon (0.063 g) in ethyl acetate (2 mL) andethanol (1 mL) was stirred under H₂ (40-50 psi) for 6 h. The mixture wasfiltered thru Celite and the filtrate concentrated to afford methyl6-(3-amino-2-methoxy)pyridylacetate (0.041 g, 100%) as a light yellowoil: ¹H NMR (CDCl₃, 300 MHz, ppm) 6.82 (d, 7.6 Hz, 1H), 6.65 (d, 7.6 Hz,1H), 3.94 (s, 3H), 3.70 (s, 3H), 3.65 (s, 2H).

D. To a solution of methyl 6-(3-amino-2-methoxy)pyridylacetate (0.078 g,0.33 mmol) and triethylamine (50 mL, 0.36 mmol) in methylene chloride(1.0 mL) was added o-tolyl isocyanate (41 μL, 0.36 mmol). The reactionwas stirred for 4 h then concentrated. The crude product was purified byflash chromatography (silica gel, 3:2 hexane-ethyl acetate) to affordthe Methyl 6-(2-methoxy-3-o-tolylureido)pyridylacetate (0.060 g, 55%) asa white powder: ¹H NMR (CDCl₃, 300 MHz, ppm) 8.33 (d, 7.9 Hz, 1H), 7.51(d, 7.8 Hz, 1H), 7.41 (s, 1H), 7.17 (m, 2H), 7.08 (m, 2H), 6.77 (d, 7.9Hz, 1H), 3.81 (s, 3H), 3.71 (s, 3H), 3.67 (s, 2H), 2.20 (s, 3H).

E. A solution of methyl 6-(2-methoxy-3-o-tolylureido)pyridylacetate(0.023 g, 0.070 mmol) in methanol (1.0 mL) was treated with 2 M LiOH (90μL, 0.18 mmol). The reaction was stirred for 18 h, diluted with H₂O (5.0mL) and washed with ether (2×). The aqueous solution was then acidifiedwith 5% aq. citric acid. The product was filtered and washed with H₂Othen ether to give 6-(2-Methoxy-3-o-tolylureido)pyridylacetic acid(0.014 g, 64%) as a white solid: ¹H NMR (CD₃OD, 300 MHz, ppm) 8.50-8.25(m, 3H), 7.60 (bd, 1H), 7.28-7.00 (m, 3H), 4.01 (s, 3H), 3.69 (s, 2H),2.30 (s, 3H); MS, m/z 316.

F. Procedure C was performed using amine β-2. The resulting product wassubjected to the conditions described in Procedure D1 to provideTFA-amine salt 1336-1.

G. The procedure described in Example 1A was performed using6-(2-methoxy-3-o-tolylureido)pyridylacetic acid (0.014 g, 0.044 mmol)and amine-TFA salt 1336-1 (0.017 g, 0.045 mmol) to afford BIO1336t-butyl ester (0.024 g, 79%) as a white foam: ¹H NMR (CDCl₃, 300 MHz,ppm) 8.40 (d, 7.9 Hz, 1H), 7.63 (d, 8.3 Hz, 1H), 7.50 (d, 7.9 Hz, 1H),7.43-7.06 (m, 6H), 6.80-6.67 (m, 4H), 5.92 (s, 2H), 5.19 (m, 1H), 4.47(m, 1H), 3.91 (s, 3H), 3.61 (s, 3H), 2.65 (m, 2H), 2.31 (s, 3H), 1.58(m, 3H), 1.31 (s, 9H).

H. To a solution of BIO1336 t-butyl ester (0.024 g, 0.035 mmol) inmethylene chloride (3.0 mL) was added trifluoroacetic acid (3.0 mL). Thereaction was stirred for 2 h then concentrated. The crude product waspurified by HPLC to afford BIO-1336 (0.011 g, 50%) as a white powder: ¹HNMR (CD₃SOCD₃, 300 MHz, ppm) 8.73 (s, 1H), 8.52 (s, 1H), 8.47 (d, 8.3Hz, 1H), 8.31 (d, 7.9 Hz, 1H), 8.11 (d, 8.3 Hz, 1H), 7.81 (d, 7.9 Hz,1H), 7.21-7.09 (m, 2H), 7.00-6.70 (m, 5H), 5.98 (s, 2H), 5.08 (m, 1H),4.36 (m, 1H), 3.97 (s, 3H), 3.52 (m, 2H), 2.64 (m, 2H), 2.25 (s, 3H),1.55-1.25 (m, 3H), 0.81 (m, 6H); HPLC (Gradient B), 20.0 min; MS, m/z620.

EXAMPLE 62 Synthesis of BIO-1382

A. To methyl 6-amino-2(S)-N-BOC-aminohexanoate hydrochloride salt (200mg, 0.60 mmol) in CH₂Cl₂ (5 ml) and TEA (basic to litmus) is addedmethanesulfonyl chloride (76.2 mg, 0.67 mmol) dropwise over 2 min. atrt. Following 1 hour of stirring the reaction is diluted with CH₂Cl₂ (10ml) partitioned 3 times with 5% citric acid (3×0.5 ml), water (1×1 ml),brine (1×1 ml), and dried over MgSO₄. The organic phase was concentratedin vacuo to yield 1382-1 (230 mg, 100%) as a clear oil.

¹HNMR(CDCl₃, 300 MHz, ppm) 7.26 (s, 5H), 5.58(d, 1H, J=8), 5.02 (s,2H),4.27 (m, 1H), 3.64 (s, 3H), 3.02 (m, 2H), 2.78 (s, 3H) 1.85-1.20 (m,6H). HPLC (Gradient 3) 24.26 min. 98% MS, mz 373.

B. To 1382-1 (225 mg, 0.60 mmol) in 10 ml MeOH at rt with stirring isadded 2N LiOH (0.91 ml, 1.8 mmol) dropwise over 2 min. Stirring iscontinued overnight. The reaction mixture is acidified with TFA (red tolitmus) and concentrated in vacuo. The clear crude gum was taken up inEtOAc (20 ml) and worked up as described in Example 62A yielding 1382-2(122 mg, 57w) as a clear gum. ¹HNMR(CDCl₃, 300 Mz, ppm), 7.33(s, 4H),5.54(d, 1H J=7.89), 4.39(m, 1H), 3.47(S, 3H), 3.09 (m, 2H), 1.92-1.28(m,6H). HPLC (Gradient 3) 19.23 min. (100%). MS, mz 359.

C. The procedure described in Example 1A was performed using 1382-2 (48mg, 0.13 mmol) and amine β-14 (25 mg, 0.09 mmol) to give 1382-3 (51 mg,62%).

¹HNMR(CDCl₃, 300 MHz, ppm), 7.97(d, 2H J=7.38), 7.35(m, 7H), 5.51(m,1H), 5.35 (dd, 1H J=5.77, 13.50), 5.09(s, 2H), 4.75(m, 1H), 4.14(m, 1H),3.88(s, 3H), 3.62(s, 3H), 3.09(m, 2H), 2.73(m, 2H), 1.92-1.77(m, 1H),1.70-1.55(m, 1H), 1.55-1.49(m, 2H), 1.49-1.15(m, 13H).

D. The CBZ protecting group of compound 1382-3 was removed undercatalytic hydrogenation conditions as described in Procedure D2 to give(13.2 mg, 35%) of product 1382-4. ¹HNMR(CDCl₃, 300 MHz, ppm).8.23-8.12(m, 2H), 8.02-7.82(m, 2H), 7.49-7.38(m, 2H), 5.50-5.31 (m, 1H),3.86(s, 3H), 3.57(s, 3H), 3.20-2.65(m, 4H), 1.89-172(m, 1H),1.50-1.10(m, 14H).

E. The procedure described in Example 49 was performed using 1382-4(15.5 mg, 0.05 mmol)to give Bio 1382 t-butyl ester (22.6 mg, 111%) as awhite solid.

¹HNMR(CDCl₃, 300 MHz, ppm). 8.02(d, 1H J=8.1), 7.87(d, 2H J=8.0),7.59(d, 1H J=8.1), 7.29-7.19(m, 5H), 7.11-7.02(m, 4H), 6.92(t, 1HJ=7.19), 5.25-5.16(m, 1H), 4.20-4.30(m, 1H), 3.8(s, 3H), 3.39 (s, 2H),2.86-2.73(m, 5H), 2.68-2.58(m, 2H), 2.17(s, 3H), 1.65-1.18(m, 15H). MS,mz 752.

F. Bio 1382 t-butyl ester (27.6 mg, 0.027 mmol) is stirred in CH₂Cl₂ (1ml) at 5° C. TFA (1.0 ml) is added in one portion; the ice bath isremoved and stirring is continued for 2 hours. The reaction mixture isconcentrated in vacuo and subjected to preparative HPLC purification toprovide BIO-1382 (14 mg, 75%) as a white solid. ¹HNMR(DMSOD₆, 300 MHz, ,ppm) 8.71(d, 1H J=7.82), 8.21(d, 1H J=8.01), 8.04-7.91 (m, 3H),7.59-7.44(m, 3H), 7.32-7.20(m, 3H), 7.01-6.98(m.2H), 5.30(dd, 1H J=7.50,14.93) 4.35(m, 1H), 3.93(s, 3H), 3.84-3.62(m, 2H), 3.09-3.45(m, 2H),2.99-2.78(m, 6H), 2.32(s, 3H) 1.75-1.15(m, 6H). HPLC (Gradient 3) 27.8min. (95%). MS, mz 696.

EXAMPLE 63 Synthesis of BIO-1400

A. To 4-phenyl-1-butene (3.47 g, 3.94 mL, 26 mmol) at RT was addedchlorosulfonyl isocynate (3.54 g, 2.17 mL, 25 mmol) under argon. Theresulting mixture was stirred overnight. The reaction mixture was addeddropwise to a rapidly stirring solution of NaHCO₃ (5 g), NaHSO₃ (1.5 g)and H₂O/CH₂Cl₂ (15 mL/10 mL) at 0° C. After 1 h, the solution wasconcentrated under reduced pressure and the residue was extracted withEtOAc (2×50 mL). After separation, the organic layer was washed withsat. NaCl (30 mL), dried with Na₂SO₄ and concentrated under reducedpressure to give 600 mg (14%) of the beta lactam 1400-1 as a lightyellow oil: ¹H NMR (CDCl₃, 300 MHz, ppm) δ 7.30-7.13 (m, 5 H, Ar), 6.45(s, 1H, NH), 3.0 (ddd, J=14.8, 4.7, 1.7 Hz, 1 H), 2.64 (t, J=7.6 Hz, 2H), 2.52 (d, J=14.8 Hz, 1H), 1.92 (m, 2 H); TLC, 50% Hex/EtOAc,R_(f)=0.27.

B. A solution of the beta lactam 1400-1 (500 mg, 2.86 mmol), MeOH (25mL), and HCl (1 mL of 33%) was stirred at RT for 18 h. The reactionmixture was diluted with EtOAc (100 mL) and basified with Et₃N untilpH=9-10 (pH paper). The resulting solution was washed with H₂O (10 mL),Sat. NaHCO₃ (30 mL), Sat. NaCl (30 mL), dried with Na₂SO₄, andconcentrated under reduced pressure to give 270 mg (52%) of amine 1400-2as a yellow oil: ¹H NMR (CDCl₃, 300 MHz, ppm) δ 7.28-7.15 (m, 5 H, Ar),3.66 (s, 3 H, OMe), 2.66 (m, 2 H), 2.48 (dd, J=15.7, 4.0 Hz, 1 H), 2.29(dd, J=15.7, 8.8 Hz, 1 H), 1.70 (m, 2 H), 1.54 (s, 2 H, NH); TLC, 10%MeOH/CH₂Cl₂, R_(f)=0.35; FABMS, m/z 207 (Cl₂H₁₇NO₂ of M⁺+1 requires207).

C. Free amine 1400-2 (100 mg, 0.55 mmol)) was coupled withNα-t-Boc-Nε-leu-N-hydroxysuccinimide (163 mg, 1.52 mmol) as described inProcedure C to give material which was deprotected with trifluoroaceticacid (0.5 mL) and then basified with Et₃N as described in Procedure D1to give the amine 1400-3 in 95% yield: ¹H NMR (CDCl₃, 300 MHz, ppm) δ9.02 (d, J=9.0 Hz, 1 H), 7.27-7.14 (m, 5 H, Ar), 4.26 (m, 1 H), 3.64 (s,two peaks, 3 H, OMe), 3.44 (m, 1 H), 2.79 (s, 2 H), 2.62 (t, J=7.8 Hz, 1H), 2.54 (d, J=4.9 Hz, 1 H), 1.87 (m, 2 H), 1.68 (m, 2 H), 1.36 (m, 1H), 0.92 (m, 6 H); TLC, 10% MeOH/CH₂Cl₂, R_(f)=0.47 and 0.18; HPLC(gradient 1) 12.2 min and 13.6 min (1:1); FABMS, m/z 321 (C₁₈H₂₈N₂O₃ ofM⁺+1 requires 321).

D. 2-Methylphenylureaphenylacetic acid (64 mg, 0.24 mmol) was coupledwith free amine 1400-3 (64 mg, 0.20 mmol) as described in Example 49 togive compound 1400-4 in 60%: ¹H NMR (DMSO-d⁶, 300 MHz, ppm) δ 9.50 (d,J=6.8 Hz, 1 H), 8.26-8.17 (m, 2 H), 7.97 (d, J=6.1 Hz, 1H), 7.84 (d,J=8.0 Hz, 1 H), 7.38 (m, 4 H), 7.27-7.09 (m, 9 H), 6.91 (t, J=7.3 Hz, 1H), 4.26 (m, 1 H), 4.03 (m, 1 H), 3.52 (s, two peaks, 3 H, OMe), 3.38(m, 2 H), 2.57-2.40 (m, 4 H), 2.25 (s, 3 H), 1.70-1.41 (m, 5 H), 0.86(m, 6 H); FABMS, m/z 587 (C₃₄H₄₂N₄O₅ of M⁺+1 requires 587).

E. Compound 1400-4 (70 mg, 0.119 mmol) in DMSO (1 mL) and MeOH (2 mL)was hydrolyzed with aqueous LiOH as described in Example 1B. The productwas purified on a Vydac reverse-phase C18 column (22 mm×25 cm) using alinear gradient of 20% CH₃CN/H₂O (0.1% TFA) to 50% CH₃CN/H₂O (0.1% TFA)with a flow rate of 10 mL/min to give the BIO-1400 in 22% isolatedyield: ¹H NMR (DMSO-d⁶, 300 MHz, ppm) δ 8.93 (m, 1 H), 8.14 (m, 1 H),7.91-7.81 (m, 3 H), 7.34 (m, 2 H), 7.27-7.09 (m, 9 H), 6.92 (t, J=7.4Hz, 1 H), 4.27 (m, 1 H), 4.00 (m, 1 H), 3.43 (d, J=14.2 Hz, 1 H), 3.36(d, J=14.2 Hz, 1 H), 2.60-2.30 (m, 4 H), 2.22 (s, 3 H), 1.68-1.55 (m, 3H), 1.45 (t, J=6.9 Hz, 2 H), 0.86 (m, 6 H); HPLC (gradient 1) 20 min and20.5 min (1:2.45); FABMS, m/z 573 (C₃₃H₄₀N₄O₅ of M⁺+1 requires 573).

Conditions for analytical HPLC:

Gradient 1: a linear gradient of 20% CH₃CN/H₂ O (0.1% TFA) to 70%CH₃CN/H₂O (0.1% TFA)

Gradient 8: a linear gradient of 15% CH₃CN/H₂O (0.1% TFA) to 40%CH₃CN/H₂O (0.1% TFA).

EXAMPLE 64 Synthesis of BIO 1051

A. 4-Aminobenzoic acid (420 mg, 3.1 mmol) in CH₂Cl₂ was treated withphenyl isocyanate (340 μl, 3.1 mmol) at RT. The reaction was stirred for20 minutes and then concentrated. The residue was washed with 1N HClthen excess ether to afford the product (98 mg, 12%) as a white powder.¹H NMR: (CDCl₃, 300 MHz, ppm), 9.08 (s, 1H), 8.80 (s, 1H), 7.90 (d, 2H),7.58 (d. 2H), 7.45 (d, 2H), 7.30 (m, 2H), 7.00 (m, 1H). FAB:257 (M+H)+,MW 256.26.

B. A solution of the amine from Example 6A (15 mg, 0.045 mmol) and theproduct from Example 64A (12 mg, 0.047 mmol) in DMF was treated withDIPEA (40 μl, 0.22 mmol) and BOP (20 mg, 0.045) at RT. After thereaction was stirred overnight it was worked up as in Example 1A toafford BIO-1051-OtBu (18 mg, 69t) as a foam.

C. BIO-1051-OtBu (19 mg, 0.031 mmol) was treated with TFA (2 mL) at RTfor 30 min and then concentrated. The crude product was purified by HPLCto afford BIO-1051 (6.3 mg, 39%) as a white powder: HPLC (gradient A)19.2 min, FAB: 517 (M+H)+, MW 516.3.

EXAMPLE 65 Synthesis of BIO-1110

A. The amine from Example 6A (49 mg, 0.15 mmol) in CH₂Cl₂ was treatedwith TFA (10 mL) at RT. The reaction was stirred for 3 hours andconcentrated. The residue was dissolved in DMF and neutralized withtriethylamine at RT. This was followed by addition of4-nitrophenylphenylisocyanate (26.5 mg, 0.16 mmol) and stirred 1 hr. atRT. Purification by HPLC resulted in 62 mg of a beige solid. ¹HNMR(CDCl₃, 300 MHz, ppm): 8.05 (d, 2H), 7.25 (m, 5H), 5.35 (m, 1H), 4.34(m, 1H), 2.22 (m, 2H), 1.59 (m, 3H), 0.84 (m, 6H). FAB: 442.9 (M+H)⁺, MW442.41. HPLC: (Gradient A) 21.05 min.

B. The product of Example 65A (55 mg, 0.12 mmol) was reduced with 10%Pd/C in MeOH while stirring under 40 psi hydrogen gas. The reactionmixture was filtered through Celite 545 and concentrated to yield 49 mgof a beige solid. ¹HNMR (CDCl₃, 300 MHz, ppm): 7.19 (m, 5H), 7.03 (d,2H), 6.94 (d, 2H), 5.27 (m, 1H), 4.23 (m, 2H), 2.72 (m, 2H), 1.52 (m,3H), 0.78 (m, 6H). FAB: 413.3 (M+H)⁺, MW 412.45. HPLC: (Gradient A)11.93 min.

C. The product of Example 65B (5 mg, 0.012 mmol) in DMF andtriethylamine was treated with phenylisocyanate (1.4 mg, 0.12 mmol).After stirring overnight, the material was purified by HPLC. ¹HNMR(CDCl3, 300 MHz, ppm): 7.55 (d, 2H), 7.36 (m, 12H), 7.04 (m, 1H), 6.34(d, 1H), 5.36 (m, 1H), 4.41 (m, 1H), 2.78 (m, 2H), 1.39 (m, 3H), 0.91(m, 6H). FAB: 532 (M+H)⁺, MW 531.36. HPLC: (Gradient A) 20.31 min.

EXAMPLE 66 Synthesis of BIO-1527

A. To a solution of amine β-3 (1 equiv.) In CH₂Cl₂ was added BOC-Pro-OSu(1 equiv.) and then stirring at rt overnight. The resulting mixture wasdiluted with ethylacetate and then washed with 5% citric acid (2×), sat.aq NaHCO3 (2×) and brine (1×), dried (Na₂SO₄), filtered and concentratedto give crude product as a white foam. The above crude product wasdissolved in CH₂Cl₂ and TFA was added at 0° C. Mixture was stirred atroom temperature for 1 hour and concentrated to give the amine as a TFAsalt.

B. To a solution of 2-methylphenylureaphenylacetic acid in DMF was addedHOBT (1.5 equiv.) and EDC (1.2 equiv.) followed by free amine fromExample 66A and then stirred at room temperature overnight. Theresulting mixture was diluted with ethylacetate and then washed with 5%citric acid (2×), sat. aq NaHCO₃ (2×) and brine (1×), dried (Na₂SO₄),filtered and concentrated to give methyl ester. The resulting methylester was dissolved in methanol and then treated with 1N LiOH (aqueoussolution). The final product (carboxylic acid) was purified by HPLC. Thepure fraction from HPLC purification was collected and dried to giveBio-1527.

Mass Spect: 573 (M +1), 595 (M +Na).

EXAMPLE 67 Inhibition of VLA4-Dependent Adhesion to BSA-CS1

This assay was used to assess the potency of VLA4-directed inhibitorycompounds of this invention.

1. Conjugation of CS1 to BSA

We dissolved BSA-SMCC (Pierce Chemical, Rockford, Ill.; Catalog # 77115)in H₂O at a concentration of 10 mg/mL. [SEQ IDNO:4]:Cys-Tyr-Asp-Glu-Leu-Pro-Gln-Leu-Val-Thr-Leu-Pro-His-Pro-Asn-Leu-His-Gly-Pro-Glu-Ile-Leu-Asp-Val-Pro-Ser-Thr(“Cys-Tyr-CS1 peptide”), which we synthesized by conventional solidphase chemistry and purified by HPLC, was dissolved in 10 mM HEPES pH 5,50 mM NaCl and 0.1 mM EDTA also at a concentration of 10 mg/mL. We thenmixed 500 μL of BSA-SMCC, 250 μL of Cys-Tyr-CS1 peptide and 75 μL of 1mM HEPES pH 7.5 and allowed the conjugation reaction to proceed for 30minutes. We stopped the reaction by adding 1 μL of beta-mercaptoethanol.Samples were analyzed for cross-linking by SDS-PAGE. This reactionproduced multiple molecules of the Cys-Tyr-CS1 peptide conjugate to eachBSA molecule.

2. Preparation of Plates for Adhesion Assay

We coated the wells of a Linbro titertek polystyrene 96-well flat bottomplate (Flow Laboratories, Maclean, Va.; catalog #76-231-05) with 100 μLof the above-described BSA-CS1 solution diluted to 1 μg/mL in 0.05 MNaHCO₃ (15 mM NaHCO₃, 35 mM Na₂CO₃) pH 9.2. Some wells were not coatedwith CS1 in order to assess non-specific cell binding (NSB). The platewas then incubated overnight at 4° C.

Following this incubation, the contents of the wells were removed byinverting and blotting the plate. All of the wells were then blockedwith 100 μL of 1% BSA in PBS, 0.02% NaN₃, for a minimum of one hour atroom temperature.

3. Preparation of Fluorescently Labelled Ramos Cells

Ramos cells are grown, maintained and labelled in RPMI 1640 culturemedium containing 1% BSA. Just prior to running the assay, we added2′,7′-bis-(2-carboxyethyl)-5 (and -6) carboxyfluorescein acetoxymethylester (“BCECF-AM”; Molecular Probes Inc., Eugene, Oreg.; catalog#B-1150) to a final concentration of 2 μM to a culture of Ramos cells(4×10⁶ cells/mL). We incubated the cells for 20 minutes at 37° C.

Following labelling, the cells were washed twice in assay buffer (24 mMTRIS, 137 mM NaCl, 2.7 mM KCl, pH 7.4, containing 0.1% BSA and 2 mMglucose) to remove any cations originating from the culture medium. Thecells were then resuspended in assay buffer to 4×10⁶ cells/mL and 2 mMMnCl₂ was added to upregulate VLA4 on the surface of the cells.

4. Running the Assay

Immediately prior to running the assay, we removed the BSA blockingsolution from the 96-well plates and washed the wells with 100 μL ofassay buffer. We then added to each well 25 μL of test compound at 2×thefinal concentration and 25 μL of the labelled Ramos cells. Finalconcentrations were selected across a range of anticipated IC50s,usually between 0.01 nM-10 μM. Each concentration of compound was testedin triplicate. The compound and cells are allowed to incubate for 30minutes at room temperature.

We then emptied the contents of the plate and washed the wells 4 timeswith assay buffer. Using a light microscope, we examined the the NSBwells. If more than a few cells are bound to those wells, we washed theplate once more to remove the excess non-specifically bound cells.

Binding of the Ramos cells to the CS1 peptide-coated wells was measuredby adding 100 μL of assay buffer to each well and quantitatingfluorescence in a Millipore Cytofluor 2300 System platereader set at 485nm excitation and 530 nm emission. Binding was expressed as an IC50—theconcentration of inhibitor at which 50% of control binding occurs.Percent binding is calculated by the formula:

[(F _(TB) −F _(NS))−(F _(I) −F _(NS))]/[(F _(TB) −F _(NS))×100=%binding,

where F_(TB) is total fluorescence bound to CS1-containing wells withoutadded inhibitor; F_(NS) is fluorescence bound in wells lacking CS1; andF_(I) is fluorescence bound in wells containing an inhibitor of thisinvention.

Other compounds according to this invention were similarly assayed. TheIC50 for each of these compounds is indicated in the table below:

BIO # IC₅₀ BIO # IC₅₀ BIO # IC₅₀ BIO # IC₅₀ 1002 nd 1064 B 1122 C 1185 A1003 nd 1065 B 1123 C 1186 B 1004 C 1066 nd 1124 nd 1187 C 1005 C 1067 B1125 nd 1188 C 1006 B 1068 B 1126 C 1189 C 1007 C 1069 A 1127 B 1190 A1008 C 1070 B 1128 B 1191 B 1009 C 1072 A 1129 B 1192 A 1010 B 1073 B1130 B 1193 B 1011 C 1074 B 1131 B 1194 A 1013 nd 1075 B 1132 B 1195 A1014 C 1076 B 1133 B 1196 A 1015 B 1077 B 1134 B 1197 A 1016 C 1078 B1135 A 1198 C 1017 C 1079 A 1136 B 1199 B 1018 C 1080 B 1137 nd 1200 B1020 C 1081 B 1138 B 1201 B 1021 B 1082 C 1139 B 1206 A 1022 C 1083 nd1140 nd 1207 C 1023 B 1084 nd 1141 nd 1208 B 1024 C 1085 C 1142 nd 1209C 1025 nd 1086 B 1143 C 1210 A 1026 C 1087 C 1144 B 1212 A 1027 C 1088 A1145 B 1214 B 1028 B 1089 A 1146 B 1215 C 1029 C 1090 A 1147 B 1216 B1030 C 1091 B 1148 C 1217 A 1031 C 1092 C 1149 C 1218 B 1032 C 1093 C1150 C 1219 B 1036 B 1094 C 1152 nd 1220 B 1037 B 1096 C 1153 C 1221 A1038 C 1097 B 1154 nd 1222 A 1039 B 1098 C 1155 nd 1223 nd 1040 B 1099 C1156 nd 1224 A 1041 nd 1100 B 1157 C 1225 nd 1042 nd 1101 C 1158 B 1227nd 1043 nd 1102 nd 1159 C 1238 A 1044 nd 1103 C 1160 B 1245 A 1045 nd1104 B 1162 nd 1246 A 1046 C 1105 B 1163 B 1248 A 1047 nd 1106 C 1164 B1270 A 1048 nd 1107 C 1168 B 1282 A 1049 nd 1108 C 1169 B 1294 A B 1050A 1109 C 1170 B 1321 A 1051 nd 1110 B 1173 B 1327 B 1052 B 1111 C 1174 B1336 A 1053 B 1112 C 1175 B 1360 A 1054 B 1113 C 1176 B 1380 B 1055 A1114 C 1177 B 1382 A 1056 A 1115 B 1178 B 1390 B 1057 nd 1116 nd 1179 A1396 B 1058 nd 1117 C 1180 B 1400 A 1060 B 1119 nd 1181 B 1272 A 1063 B1120 nd 1182 B 1311 B 1319 B 1345 A 1347 A 1358 B 1361 A 1388 A 1393 A1429 B 1444 B 1474 B 1475 B 1490 A 1515 A 1525 B 1526 B 1536 A 1594 B1648 B 1655 B 1721 B 1725 nd 1726 nd 1727 nd 1728 nd 1729 nd 1730 nd1731 nd 1732 nd Table abbreviations: A - <50 nM; B - 50 nM - 10 μM;C - >10 μM; nd - not determined. All compounds tested in this tabledemonstrated an IC₅₀ < 1 mM

EXAMPLE 68 Direct Binding Of VLA4-Presenting Cells To VCAM-IgG

We next examined the ability of the compounds of this invention toinhibit VCAM/VLA4 binding, utilizing a VCAM-IgG-alkaline phosphataseconjugate. To carry out this assay, we used the Millipore MultiscreenAssay System (Millipore Corp., Bedford, Mass.) to wash the cellsefficiently.

1. Preparation of VCAM-IgG-AP Conjuagtes

The construction of VCAM 2D-IgG expression vectors, transfection of CHOcells with those constructs and purification of the resulting expressionproduct is described in PCT publication WO 90/13300, the disclosure ofwhich is herein incorporated by reference.

1.2 ml of purified VCAM 2D-IgG (5 mg/ml in 10 mM HEPES, pH 7.5) wasreacted with 44 μl of Traut's reagent (2-iminothiolane, 20 mg/ml inwater; Pierce Chemical, Rockford, Ill.) at room temperature for 30minutes. The sample was desalted on a 15 ml Sephadex G-25 columnequilibrated with 100 mM NaCl, 10 mM MES, pH 5.0. One ml fractions werecollected and absorbance at 280 nm was determined. The two peakfractions were pooled.

One ml of calf intestinal alkaline phosphatase (19 mg/ml; PierceChemical, Rockford, Ill.) was reacted with 100 μl of sulfo-SMCC (30mg/ml in water) and 100 μl 1 M HEPES, pH 7.5 for 35 minutes at roomtemperature. The reaction mix was desalted on a 12 ml Sephadex G-25column equilibrated with 150 mM NaCl, 10 mM HEPES, pH 6.0. One mlfractions were collected and absorbance at 280 nm was determined. Thetwo peak fractions were pooled and stored on ice.

The alkaline phosphatase-SMCC and VCAM 2D-IgG-iminothilane adducts werecross-linked at a molar ratio of 2:lin Tris-HCL, pH 7.5 by incubation atroom temperature for 30 minutes. Extent of cross-linking was determinedby SDS-PAGE. The cross-linked products were stabilized by the additionof 2 mM MgCl₂ and 0.25 nM ZnCl₂ and stored at 4° C.

2. Binding Assay

We first blocked a 96-well filtration plate for by adding 275 μL of PBScontaining 0.1% Tween 20 and 2% BSA (“blocking buffer”) to each well andincubating for 1 hour at room temperature. The plate was then placedonto a vacuum manifold and the blocking buffer was drained through thebottom of the filtration wells into a waste collection tray. Then wewashed the wells three times with 200-250 μL of Tris-buffered saline,containing 0.1% BSA, 2 mM glucose and 1 mM HEPES, pH 7.5 (“assaybuffer”) to wash out any remaining blocking buffer. We then drained theplates and blotted them on paper towels to remove buffer on theunderside of the plate.

We then prepared a stock solution of VCAM-IgG-AP (4 μg/mL in assaybuffer) and filtered it thorugh a 0.2μ low protein binding syringefilter (Gelman Sciences, Ann Arbor, Mich. # 4454). This solution wasthen diluted 1:10 in assay buffer and 25 μL was added to every well ofthe washed plate.

We diluted the cell adhesion inhibitor being tested to 2× finalconcentration in assay buffer and added 25 μL of each dilution totriplicate wells in the plate. Final concentrations used ranged from0.01 nM-10 μM. Control wells for total binding and non-specific bindingrecieved 25 μL of assay buffer, instead of inhibitor. Total bindingwells contained cells and VCAM-IgG-AP in assay buffer. Non-specificbinding wells contained only VCAM-IgG-AP in assay buffer.

Jurkat cells were washed once in assay buffer to remove growth mediumand resuspended at 8×10⁶/mL in assay buffer containing 2 mM MnCl₂. Weadded 50 μl of Jurkat cells to every well, except the non-specificbinding wells, which received 50 μL of assay buffer to maintain a finalassay volume of 100 μL per well. We gently mixed the contents of thewells by tapping the sides of the plate. The plate was then allowed toincubate undisturbed for 60 minutes at room temperature.

At the end of the 60 minute incubation, we placed the plate on thevacuum manifold to drain the wells. We carefully added 100 μL of assaybuffer containing 1 mM MnCl₂ (wash buffer) to each well so as not todisturb the cells on the bottom. The wash buffer was removed by vacuumand the plate was washed again with 150 μL of wash buffer. Afterdraining the wash buffer again, the underside of the plate was blottedon paper towels.

Next, we prepared a 10 mg/mL solution of 4-nitrophenylphosphate in 0.1 Mglycine, 1 mM ZnCl₂, pH 10.5 (substrate buffer) and added 100 μLimmediately added to each well. The plate was incubated for 30 minutesat room temperature to allow the calorimetric reaction to proceed. Westopped the reaction by adding 100 μL of 3 N NaOH to each well.

The contents of the 96-well filtration plate was then transferreddirectly into a 96-well flat bottom plate using the vacuum manifold. Theplate was read at a wavelength of 405 nm to determine the amount of VCAMconjugate bound to the cells. Percent binding is calcualted by theformula:

[(A _(TB) −A _(NS))−(A _(I) −A _(NS))]/[(A _(TB) −A _(NS))×100=%binding,

where A_(TB) is the absorbance at 405 nm of CS1-containing wells withoutadded inhibitor; A_(NS) is the absorbance at 405 nm in wells lackingCS1; and A_(I) is absorbance at 405 nm in wells containing an inhibitorof this invention

We assayed other compounds of this invention in the same assay. The IC50values are comparable to those derived from the CS1 binding assaydescribed in the previous example, although certain compoundsdemonstrated up to 10-fold greater binding in this assay than in theprevious assay.

EXAMPLE 69 Inhibition Of Mouse Contact Hypersensitivity

We anesthetized 20-g female Balb/c mice (Jackson Laboratories, BarHarbor, Me.) with sodium pentobarbital (90 mg/kg, i.p.). A 3 cm² patchof abdominal skin was then exposed by closely shaving the fur. The skinwas then scrubbed with 70% ethanol, followed by application of 25 μL of0.5% DNFB in 4:1 v/v acetone:olive oil onto the bare abdominal skin. Wethen lightly scratched the skin with the applying pipet tip to encouragemild inflammation. Twenty four hours after the initial sensitization weagain sensitized the mouse with 25 μL of 0.5% DNFB at same abdominalskin location, again followed by light scratching with the pipet tip.The second sensitization was performed while restraining theunanesthetized mouse.

On Day 5 (120 hours after the initial sensitization), we anesthetizedthe mice with 90:10 mg/kg ketamine:xylazine, i.p. and applied asub-irritant dose of 10 μL of 0.2% DNFB to the dorsal surface of theleft ear. The right ear received a similar application of the 4:1 v/vacetone:olive oil vehicle.

Four hours after challenging the immune response, we administeredvarious concentrations of the inhibitors of this invention to the micein 100 μL 0.5% sodium phosphate buffer, pH 8.8, and 3% v/v DMSO bysubcutaneous (s.c.) injection. Less soluble inhibitors occasionallyrequired up to 30% DMSO addition the highest concentrations tested.Groups of 8 mice were used for each treatment tested. Positive (PS2anti-mouse VLA-4 antibody, 8 mg/kg, i.v.), and negative control(phosphate-buffered physiological saline, PBS, 100 μL i.v.; DMSO in PBS,100 μL s.c.) groups were routinely tested for comparison as part of theassay of test compounds.

Twenty four hours after challenge mice were again anesthetized withketamine:xylazine and the ear thickness of both ears measured with anengineer's micrometer to an accuracy of 10⁻⁴ inches. The ear swellingresponse for each mouse was the difference between its control- andDNFB-challenged ear thickness. Typical uninhibited ear swellingresponses were 65-75×10⁻⁴ in. Inhibition of the ear swelling responsewas judged by comparison of treated groups with their negative controlgroup. Percent inhibition was calculated as:$\left\lbrack \frac{\begin{matrix}\left( {{mean}\quad {negative}{\quad \quad}{control}} \right. \\\left. {{group}{\quad \quad}{ear}{\quad \quad}{swelling}} \right)\end{matrix} - \begin{matrix}\left( {{mean}\quad {test}{\quad \quad}{group}} \right. \\\left. {{ear}{\quad \quad}{swelling}} \right)\end{matrix}}{{mean}\quad {negative}\quad {control}{\quad \quad}{group}\quad {ear}\quad {swelling}} \right\rbrack \cdot 100$

Statistical significance of the difference among treatment groups wasevaluated using one-way analysis of variance followed by computation ofthe Tukey-Kramer Honestly Significant Difference (JMP, SAS Institute)using p<0.05.

The inhibitors of this invention cause a statistically significantreduction in the ear swelling response of DNFB-treated mice as comparedto uninhibited control animals.

EXAMPLE 70 Inhibition Of Ascaris Antigen-Induced Late Phase AirwaySensitivity In Allergic Sheep

Sheep which had previously been shown to develop both early and latebronchial responses to Ascaris suum antigen were used in this study. Theprotocol used for the experiment was that described in W. M. Abraham etal., J. Clin. Invest., 93, pp. 776-87 (1994), except that the VLA-4inhibitors of this invention were administered to the animals wasdissolved in 3-4 ml of 50% aqueous ethanol and delivered by aerosolspray.

The results showed that all of the VLA-4 inhibitors of this inventioninhibited the airway responses associated with administration of Ascarissuum antigen.

While we have hereinbefore presented a number of embodiments of thisinvention, it is apparent that our basic construction can be altered toprovide other compounds and methods which utilize the compounds of thisinvention. Therefore, it will be appreciated that the scope of thisinvention is to be defined by the claims appended hereto rather than thespecific embodiments which have been presented hereinbefore by way ofexample.

                   #             SEQUENCE LISTING<160> NUMBER OF SEQ ID NOS: 5 <210> SEQ ID NO 1 <211> LENGTH: 8<212> TYPE: PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: Synthetically generated pept #ide<400> SEQUENCE: 1 Glu Ile Leu Asp Val Pro Ser Thr  1               5<210> SEQ ID NO 2 <211> LENGTH: 5 <212> TYPE: PRT<213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: Synthetically generated pept #ide<400> SEQUENCE: 2 Glu Ile Leu Asp Val  1               5<210> SEQ ID NO 3 <211> LENGTH: 5 <212> TYPE: PRT<213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: Synthetically generated pept #ide<400> SEQUENCE: 3 Leu Asp Val Pro Ser  1               5<210> SEQ ID NO 4 <211> LENGTH: 27 <212> TYPE: PRT<213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: Synthetically generated pept #ide<400> SEQUENCE: 4 Cys Tyr Asp Glu Leu Pro Gln Leu Val Thr Le#u Pro His Pro Asn Leu  1               5   #                10  #                15 His Gly Pro Glu Ile Leu Asp Val Pro Ser Th #r            20       #            25 <210> SEQ ID NO 5 <211> LENGTH: 5<212> TYPE: PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: Synthetically generated pept #ide<221> NAME/KEY: VARIANT <222> LOCATION: 4<223> OTHER INFORMATION: Xaa = 4-thioproline <400> SEQUENCE: 5Arg Cys Asp Xaa Cys  1               5

We claim:
 1. A cell adhesion inhibitory compound selected from acompound of the formula (I):

or a pharmaceutically acceptable derivative of (I), wherein: X is —CO₂H;Y is selected from the group consisting of —CO—, —SO₂— and —PO₂—; R₁ isselected from the group consisting of alkenyl, alkynyl, cycloalkyl,aryl-fused cycloalkyl, cycloalkenyl, aryl, substituted aralkyl,aryl-substituted alkenyl, aryl-substituted alkynyl,cycloalkyl-substituted alkyl, cycloalkenyl-substituted alkyl, biaryl,alkenoxy, alkynoxy, aralkoxy, aryl-substituted alkenoxy,aryl-substituted alkynoxy, alkylainino, alkenylamino, alkynylamino,aryl-substituted alkylamino, aryl-substituted alkenylamino,aryl-substituted alkynylamino, aryloxy, aryl-substituted arylamino,N-alkylurea-substituted alkyl, N-arylurea-substituted alkyl,aminocarbonyl-substituted alkyl, carboxylalkyl substituted aralkyl, andoxocarbocyclyl-fused aryl; R₂ is selected from the group consisting ofhydrogen, aryl, alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, andaryl-substituted alkyl; R₃ is selected from the group consisting ofalkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, aralkyl,aryl-substituted alkenyl or alkynyl, cycloalkenyl, aralkyl,aryl-substituted alkenyl, aryl-substituted alkynyl, hydroxy-substitutedalkyl, alkoxy-substituted alkyl, aralkoxy-substituted alkyl,amino-substituted alkyl, (aryl-substitutedalkyloxycarbonylamino)-substituted alkyl, thiol-substituted alkyl,alkylsulfonyl-substituted alkyl, (hydroxy-substitutedalkylthio)-substituted alkyl, thioalkoxy-substituted alkyl,acylamino-substituted alkyl, alkylsulfonylamino-substituted alkyl,arylsulfonylamino-substituted alkyl, N-(alkyl, alkenyl or alkynyl)- orN,N-(dialkyl, dialkenyl, or dialkynyl)- orN,N-(alkyl,alkenyl)-aminocarbonyl-substituted alkyl,carboxyl-substituted alkyl, dialkylamino-substituted acylaminoalkyl andamino acid side chains selected from the group consisting of arginine,asparagine, glutamine, S-methyl cysteine, methionine and correspondingsulfoxide and sulfone derivatives thereof, glycine, leucine, isoleucine,allo-isoleucine, tert-leucine, norleucine, phenylalanine, tyrosine,alanine, ornithine, glutamine, valine, threonine, seine, a-cyanoalanine,and allothreonine; R₄ is selected from the group consisting of aryl,alkyl, cycloalkyl, alkenyl, cycloalkenyl, alkynyl, aryl-substitutedalkyl, hydrogen, amido, mono- or dialkylaminocarbonyl, mono- ordiarylaminocarbonyl, alkylarylaminocarbonyl, diarylaminocarbonyl, mono-or diacylaminocarbonyl, aromatic acyl, alkyl optionally substituted bysubstituents selected from the group consisting of amino, carboxy,hydroxy, mercapto, mono- or dialkylamino, mono- or diarylamino,alkylarylamino, diarylamino, mono- or diacylamino, alkoxy, alkenoxy,aryloxy, thioalkoxy, thioalkenoxy, thioalkynoxy, and thioaryloxy; and nis 0, 1, or 2; provided that at least one of R₁-R₄ contains an arylgroup and further provided that when Y is SO₂, R₄ is aryl, alkyl,cycloalkyl, alkenyl, alkynyl, aryl-substituted alkyl, or hydrogen, and nis 0, R₁ is not aryl, alkenyl, alkynyl, cycloalkyl, substituted aralkyl,aryl-substituted alkenyl, aryl-substituted alkynyl, orcycloalkyl-substituted alkyl.
 2. The compound according to claim 1,wherein: R₁ is selected from the group consisting of alkenyl, alkynyl,cycloalkyl, aryl-fused cycloalkyl, cycloalkenyl, aryl, substitutedaryl-substituted alkyl (“aralkyl”), aryl-substituted alkenyl or alkynyl,cycloalkyl-substituted alkyl, cycloalkenyl-substituted cycloalkyl,biaryl, alkenoxy, alkynoxy, aryl-substituted alkoxy (“aralkoxy”),aryl-substituted alkenoxy, aryl-substituted alkynoxy, alkylamino,alkenylamino or alkynylamino, aryl-substituted alkylamino,aryl-substituted alkenylamino, aryl-substituted alkynylamino, aryloxy,aryl-substituted arylamino, N-alkylurea-substituted alkyl,N-arylurea-substituted alkyl, and aminocarbonyl-substituted alkyl; R₂ isselected from the group consisting of hydrogen, aryl, alkyl, alkenyl,alkynyl, cycloalkyl, cycloalkenyl, and aryl-substituted alkyl; R₃ isselected from the group consisting of alkyl, alkenyl, alkynyl,cycloalkyl, cycloalkenyl, aralkyl, aryl-substituted alkenyl or alkynyl,hydroxy-substituted alkyl, alkoxy-substituted alkyl,aralkoxy-substituted alkyl, amino-substituted alkyl, (aryl-substitutedalkyloxycarbonylamino)-substituted alkyl, thiol-substituted alkyl,alkylsulfonyl-substituted alkyl, (hydroxy-substitutedalkylthio)-substituted alkyl, thioalkoxy-substituted alkyl,acylamino-substituted alkyl, alkylsulfonylamino-substituted alkyl,arylsulfonylamino-substituted alkyl, N-(alkyl, alkenyl or alkynyl)- orN,N-(dialkyl, dialkenyl, dialkynyl)- orN,N-(alkyl,alkenyl)-aminocarbonyl-substituted alkyl,carboxyl-substituted alkyl, and amino acid side chains selected fromarginine, asparagine, glutamine, S-methyl cysteine, methionine andcorresponding sulfoxide and sulfone derivatives thereof, glycine,leucine, isoleucine, allo-isoleucine, tert-leucine, norleucine,phenylalanine, tyrosine, alanine, ornithine, glutamine, valine,threonine, serine, beta-cyanoalanine, and allothreonine; R₄ is selectedfrom the group consisting of aryl, alkyl, cycloalkyl, alkenyl,cycloalkenyl, alkynyl and aryl-substituted alkyl; n is 0, 1, or
 2. 3.The cell adhesion inhibitory compound according to claim 1, wherein R₁is an aryl-substituted C₁-C₄ alkyl group.
 4. The cell adhesioninhibitory compound according to claim 3, wherein R₁ is a(N-Ar′-urea)-para-substituted arylalkyl group.
 5. The cell adhesioninhibitory compound according to claim 4, wherein R₁ is a(N-Ar′-urea)-para-substituted phenylmethyl group.
 6. The cell adhesioninhibitory compound according to claim 1, wherein R₁ is selected fromthe group consisting of cyclohexylmethyl, N-phenylamino, phenyl,phenylcarbonyl, phenylmethyl, t-butylamino, 1-indanyl, 1-naphthylmethyl,1-phenylcyclopropyl, 2-(4-hydroxy-phenyl)ethyl,2-(benzyloxycarbonylamino) phenylmethyl,2-(bis(phenylsulfonyl)amino)-phenylmethyl,2-(N′-phenylurea)phenylmethyl, 2-aminophenylmethyl,2-benzamidophenylmethyl, 2-bromo-4-hydroxy-5-methoxyphenylmethyl,2-hydroxyphenylmethyl, 2naphthylmethyl, 2-phenylethyl,2-[4-(N′-phenylurea)phenyl]-ethyl,3-(benzyloxycarbonylamino)-phenylmethyl, 2-(N′-phenyl-urea)phenylmethyl,2-(N′-phenylurea)propyl, 3-(phenylsulfonamido)-phenylmethyl,3-acetamidophenylmethyl, 3-aminophenylmethyl, 3-benzamidophenylmethyl,3-hydroxy-4-(N′-phenylurea)-phenylmethyl, 3-hydroxyphenylmethyl,3-methoxy-4-(N′-phenylurea)-phenylmethyl,3-methoxy-4-(N′-(2-methylphenyl)-urea)phenylmethyl,3-methyl-4-(N′-phenylurea)-phenylmethyl, 3-nitrophenylmethyl,3-phenylpropyl, 4-(2-aminobenzamido)-phenylmethyl,4-(benzamido)phenyl-methyl, 4-(benzyloxycarbonylamino)-phenylmethyl,4-(N′-(2-chlorophenyl)urea)-phenylmethyl, 4-(N′-(2-chlorophenyl)urea)-3-methoxyphenylmethyl, 4-(N′-(2-ethylphenyl)urea)phenylmethyl,4-(N′-(2-isopropylphenyl)urea)phenylmethyl,4-(N′-(2-methoxyphenylurea)-phenyl-methyl,4-(N′-(2-methyl-3-pyridyl)urea)-phenylmethyl,4-(N′-(2-nitrophenyl)urea)-phenylmethyl,4-(N′-(2-t-butylphenyl)-urea)-phenylmethyl,4-(N′-(3-chlorophenyl)urea)-phenylmethyl,4-(N′-(3-methoxyphenyl)urea)-phenylmethyl,4-(N′-(3-methylphenyl)urea)-phenylmethyl,4-(N′-(2-methylphenyl)urea)-phenylmethyl, 4-(N′-benzylurea)phenylmethyl,4-(N′-cyclohexylurea)-phenylmethyl, 4-(N′-ethylurea)phenylmethyl,4-(N′-isopropylurea)-phenylmethyl, 4-(N′-methylurea)-phenylmethyl,4-(N′-p-toluylurea)phenylmethyl, 4-(N′-phenylurea)phenyl,4-(N′phenylurea)phenylamino, 4-(N′-phenylurea)phenyl-methyl,4-(N′-t-butylurea)-phenylmethyl,4-(phenylaminocarbonylamino-methyl)-phenyl,4-(phenylsulfonamido)phenylmethyl,4-(t-butoxycarbonyl-amino)-phenylmethyl, 4-acetamidophenylmethyl,4-aminophenylamino, 4-aminophenylmethyl, 4-benzamidophenylmethyl,4-chlorophenylmethyl, 4-hydroxy-3-nitrophenylmethyl,4-hydroxyphenylmethyl, 4-methoxyphenylmethyl, 4-nitrophenylamino,4-nitrophenylmethyl, 4-phenacetamidophenylmethyl, 4-phenylphenylmethyl,4-trifluoromethylphenylmethyl,4-[2-(N′-methylurea)-benzamido]-phenylmethyl,4-(N′-(2-methylphenyl)urea)-phenylmethyl,4-(N′-phenyl-N″-methylguanidino)-phenylmethyl, 5-(N′phenylurea)pentyl,5-(N′-t-butylurea)pentyl, 2,2-dimethylpropyl, 2,2-diphenylmethyl,2,3-benzocyclobutyl, 3,4-dihydroxyphenylmethyl,3,5-dimethoxy-4-hydroxyphenylmethyl, 3-carboxy-1-phenylpropyl,3-hydroxy-4-(2-methylphenyl)ureaphenylmethyl,3-hydroxy-4-(2-chlorophenyl)ureaphenylmethyl, 6-(phenylurea)heptyl,4-phenylurea)butyl, 2-thienylmethyl,4-(2,6-dimethylphenylurea)phenylmethyl,4-(2-hydroxyphenylurea)phenylmethyl,3-butoxy-4-(2-methylphenyl)ureaphenylmethyl,3-butoxy-4-(phenylurea)phenylmethyl, 2-phenylethynyl,3-nitro-4(phenylurea)phenylmethyl,3-acylamino-4-(phenylurea)phenylmethyl, 4-(N,N-phenyl,methylurea)phenylmethyl, 4-(3-hydroxyphenylurea)phenylmethyl,4-(2-acetylaminophenylurea)phenylmethyl,4-(2-propionylaminophenylurea)phenylmethyl, 4-(N,N′-phenyl,methylurea)phenylmethyl, 4-(2-dimethylaminophenylurea)phenylmethyl,1,3-dimethyl-3-(phenylurea)butyl, hydroxyethylthiomethyl,4-(phenylurea)phenylethenyl, 3-amino-4-(phenylurea)phenylmethyl,4-(4-hydroxyphenylurea)phenylmethyl, 4-(2-aminophenylurea)phenylmethyl,4-((2-methylurea)phenylurea)phenyl,4-(2-hydroxyphenylurea)-3-methoxyphenylmethyl,4-(2-methylsulfonylmethylphenylurea)phenylmethyl,4-(2-trifluoromethylphenylurea)phenylmethyl,4-(phenylthiourea)phenylmethyl, 4-(benzyloxyurea)phenylmethyl,4-benzoylureaphenylmethyl, hydroxylureaphenylmethyl, N′,N′-methyl,hydroxylureaphenylmethyl, 4-(N′-allylurea)phenylmethyl,4-(propylurea)phenylmethyl, 4-(methoxyurea)phenylmethyl,4-(dimethylurea)phenylmethyl,4-(2-hydroxy-6-methylphenylurea)phenylmethyl,4-(3-hydroxy-2-methylphenylurea)phenylmethyl,4-(2-fluorophenylurea)phenylmethyl, 4-(3-fluorophenylurea)phenylmethyl,4-(4-fluorophenylurea)phenylmethyl,4-(2,3-dimethylphenylurea)phenylmethyl,4-(2,5-dimethylphenylurea)phenylmethyl,4-(2-methyl-4-fluorophenylurea)phenylmethyl,4-(2-methyl-3-fluorophenylurea)phenylmethyl, 3-carboxy-3-phenylpropyl,4-(5-hydroxy-2-methylphenylurea)phenylmethyl,4-(4-hydroxy-2-methylphenylurea)phenylmethyl,4-(2,4-difluorophenylurea)phenylmethyl,4-(phenoxycarbonylamino)phenylmethyl, 3-phenylureapropyl,4-(phenylaminocarbonyloxy)phenylmethyl, 4-cinnamoylphenylmethyl,4-(2-methylphenylaminocarbonloxy)phenylmethyl,methylphenylurea)phenylamino, 4-(phenylaminocarbonyl)phenylmethyl,4-phenylalkynylphenylmethyl, 3-carboxy-3-phenylpropyl, and3-methoxy-4-(phenylcarbamoyloxy)phenylmethyl.
 7. The cell adhesioninhibitory compound according to claim 1, wherein R₁ is selected fromthe group consisting of cyanomethyl, cyclohexylmethyl, N-phenylamino,phenyl, phenylcarbonyl, phenylmethyl, t-butylamino, 1-indanyl,1-naphthylmethyl, 1-phenylcyclopropyl, 2-(4-hydroxy-phenyl)ethyl,2-(benzyloxycarbonylamino)phenylmethyl,2-(bis(phenylsulfonyl)amino)-phenylmethyl,2-(N′-phenylurea)phenylmethyl, 2-aminophenylmethyl,2-benzamidophenylmethyl, 2-bromo-4-hydroxy-5-methoxyphenylmethyl,2-hydroxyphenylmethyl, 2naphthylmethyl, 2-phenylethyl,2-[4-(N′-phenylurea)phenyl]-ethyl,3-(benzyloxycarbonylamino)-phenylmethyl, 3-(N′-phenyl-urea)phenylmethyl,3-(N′-phenylurea)propyl, 3-(phenylsulfonamido)-phenylmethyl,3-acetamidophenylmethyl, 3-aminophenylmethyl, 3-benzamidophenylmethyl,3-hydroxy-4-(N′-phenylurea)-phenylmethyl, 3-hydroxyphenylmethyl,3-methoxy-4-(N′-phenylurea)-phenylmethyl,3-methoxy-4-(N′-(2-methylphenyl)-urea)phenylmethyl,3-methyl-4-(N′-phenylurea)-phenylmethyl, 3-nitrophenylmethyl,3-phenylpropyl, 4-(2-aminobenzamido)-phenylmethyl,4-(benzamido)phenyl-methyl, 4-(benzyloxycarbonylamino)-phenylmethyl,4-(N′-(2-chlorophenyl)urea)-phenylmethyl, 4-(N′-(2-chlorophenyl)urea)-3-methoxyphenylmethyl, 4-(N′-(2-ethylphenyl)urea)phenyhnethyl,4-(N′-(2-isopropylphenyl)urea)-phenylmethyl,4-(N′-(2-methoxyphenyl)urea)-phenyl-methyl,4-(N′-(2-nitrophenyl)urea)-phenylmethyl,4-(N′-(2-t-butylphenyl)-urea)-phenylmethyl,4-(N′-(3-chlorophenyl)urea)-phenylmethyl,4-(N′-(3-methoxyphenyl)urea)-phenylmethyl,4-(N′-(3-methylphenyl)urea)-phenylmethyl,4-(N′-(2-methylphenyl)urea)-phenylmethyl, 4-(N′-benzylurea)phenylmethyl,4-(N′-cyclohexylurea)-phenylmethyl, 4-(N′-ethylurea)phenylmethyl,4-(N′-isopropylurea)-phenylmethyl, 4-(N′-methylurea)-phenylmethyl,4-(N′-p-toluylurea)phenylmethyl, 4-(N′-phenylurea)phenyl,4-(N′-phenylurea)phenylamino, 4-(N′-phenylurea)phenyl-methyl,4-(N′-t-butylurea)-phenylmethyl,4-(phenylaminocarbonylamino-methyl)-phenyl,4-(phenylsulfonamido)phenylmethyl,4-(t-butoxycarbonyl-amino)-phenylmethyl, 4-acetamidophenylmethyl,4-aminophenylamino, 4-aminophenylmethyl, 4-benzamidophenylmethyl,4-chlorophenylmethyl, 4-hydroxy-3-nitrophenylmethyl,4-hydroxyphenylmethyl, 4-methoxyphenylmethyl, 4-nitrophenylamino,4-nitrophenylmethyl, 4-phenacetamidophenylmethyl, 4-phenylphenylmethyl,4-trifluoromethylphenylmethyl,4-[2-(N′-methylurea)-benzamido]-phenylmethyl,4-(N′-(2-methylphenyl)urea)-phenylmethyl,4-(N′-phenyl-N″-methylguanidino)-phenylmethyl, 5-(N′-phenylurea)pentyl,5-(N′-t-butylurea)pentyl, 2,2-dimethylpropyl, 2,2-diphenylmethyl,2,3-benzocyclobutyl, 3,4-dihydroxyphenylmethyl, and3,5-dimethoxy-4-hydroxyphenylmethyl.
 8. The cell adhesion inhibitorycompound according to claim 6, wherein R₁ is selected from the groupconsisting of 4-hydroxyphenylmethyl,3-methoxy-4-(N-phenylurea)-phenylmethyl, 4-(N′-phenylurea)-phenylmethyl,4-(N′-(2-methylphenyl)urea)-phenylmethyl, and3-methoxy-4-(N′-(2-methylphenyl)urea)-phenylmethyl.
 9. The cell adhesioninhibitory compound according to claim 8, wherein R₁ is selected fromthe group consisting of 4-hydroxyphenylmethyl,3-methoxy-4-(N′-phenylurea)-phenylmethyl,4-(N′-phenylurea)-phenylmethyl,4-(N′-(2-methylphenyl)urea)-phenylmethyl, and3-methoxy-4-(N′-(2-methylphenyl)urea)-phenylmethyl.
 10. The celladhesion inhibitory compound according to claim 1, wherein Y is acarbonyl group.
 11. The cell adhesion inhibitory compound according toclaim 1, wherein R₂ is hydrogen, methyl, or phenacyl.
 12. The celladhesion inhibitory compound according to claim 11, wherein R₂ ishydrogen.
 13. The cell adhesion inhibitory compound according to claim1, wherein R₃ is selected from the group consisting of2-(methylsulfonyl)-ethyl, 3-(hydroxy-propylthio)-methyl,4-(methylsulfonylamino)-butyl, 4-acetylaminobutyl, aminomethyl, benzyl,butyl, hydroxymethyl, isobutyl, methyl, methylthiomethyl, phenylmethyl,propyl, 4-(benzloxycarbonylamino)-butyl, N,N-(methylpropargyl)amino,2-(methylthio)-ethyl, 2-(N,N-dimethylamino)ethyl, 4-amino-butyl,4-benzyloxyphenylmethyl, 2-benzylthiomethyl,t-butoxy-carbonylaminomethyl, sec-butyl, t-butyl,N,N-dimethyl-aminocarbonylmethyl, 1,1-ethano, 4-hydroxyphenylmethyl,1-hydroxyethyl, 1-methoxyethyl, 4-methoxyphenylmethyl, benzyloxymethyl,benzylthiomethyl, carbonylmethyl, 2-methylsulfinylethyl, 2-phenylethyl,asparagine side-chain, 4-(phenylurea)butyl, 4-(methylurea)butyl,4-methylsulfonylaminobutyl, hydroxymethylthiomethyl,2-methylsulfonylethyl, 4-propionylaminobutyl,4-ethoxycarbonylaminobutyl, methoxycarbonylaminobutyl,carbomethoxymethyithiomethyl, 4-t-butylureabutyl,carboxymethyithiomethyl, dimethylamidomethylthiomethyl,acetylaminopropyl, 3-methylureapropyl, 4-trifluoroacetylaminobutyl,dimethylaminomethylthiomethyl, dimethylaminoethylthiomethyl, and4-(dimethylaminoacetylamino)butyl.
 14. The cell adhesion inhibitorycompound according to claim 13, wherein R₃ is selected from the groupconsisting of 2-(methylsulfonyl)-ethyl, 3-(hydroxy-propylthio)-methyl,4-(methylsulfonylamino)-butyl, 4-acetylaminobutyl, aminomethyl, benzyl,butyl, hydroxymethyl, isobutyl, methyl, methylthiomethyl, phenylmethyl,propyl, 4-(benzyloxycarbonylamino)-butyl, N,N-(methylpropargyl)amino,2-(methylthio)-ethyl, 2-(N,N-dimethylamino)ethyl, 4-amino-butyl,4-benzyloxyphenylmethyl, 2-benzylthiomethyl,t-butoxy-carbonylaminomethyl, sec-butyl, t-butyl,N,N-dimethyl-aminocarbonylmethyl, 1,1-ethano, 4-hydroxyphenylmethyl,1-hydroxyethyl, 1-methoxyethyl, 4-methoxyphenylmethyl, benzyloxymethyl,benzylthiomethyl, carbonylmethyl, 2-methylsulfinylethyl, 2-phenylethyl,and asparagine side-chain.
 15. The cell adhesion inhibitory compoundaccording to claim 13, wherein R₃ is selected from the group consistingof isobutyl, 2-(methylthio)-ethyl, 3-(hydroxypropylthio)-methyl,2-(methylsulfonyl)-ethyl, 4-acetylamino-butyl,4-(methylsulfonylamino)-butyl, and 4-(ethoxycarbonylamino)butyl.
 16. Thecell adhesion inhibitory compound according to claim 15, wherein R₃ isselected from the group consisting of isobutyl, 2-(methylthio)-ethyl,3-(hydroxypropylthio)-methyl, 2-(methylsulfonyl)-ethyl,4-acetylamino-butyl, and 4-(methylsulfonylamino)-butyl.
 17. The celladhesion inhibitory compound according to claim 1, wherein R₄ isselected from the group consisting of 4-carbomethoxy-phenyl,4-carboxyphenyl, 4-fluorophenyl, 4-methoxy-phenyl, benzyl, methyl,phenyl, phenylmethyl, phenylethyl, 4-chlorophenyl, 3,4-difluorophenyl,3,4-dimethoxyphenyl, 2-methoxyphenyl, 3-methoxyphenyl, 4-methoxyphenyl,2-nitrophenyl, 4-phenoxyphenyl, 4-ethoxyphenyl, 4-nitrophenyl,4-acetylaminophenyl, 4-methylureaphenyl, 2-fluorophenyl, naphthyl,3-fluorophenyl, 3-nitrophenyl, hydrogen, 2-nitrophenyl, 4-cyanophenyl,3-methoxyphenyl, 4-methylsulfonylamino, 3-cyanophenyl, 4-propionylamino,4-aminophenyl, 3-aminophenyl, 4-trifluoromethoxyphenyl, 4-methylphenyl,4-amino-3-nitrophenyl, 4-hydroxy-3-methoxyphenyl, 4-hexyloxyphenyl,4-methylthiophenyl, 4-dimethylaminophenyl, 3-hydroxy-4-nitrophenyl,n-pentyl, carboxymethyl, 2-carboxyethyl, ethynyl, 2-propenyl,2-propynyl, methyl, and propyl.
 18. The cell adhesion inhibitorycompound according to claim 17, wherein R₄ is selected from the groupconsisting of 4-carbomethoxy-phenyl, 4-carboxyphenyl, 4-fluorophenyl,4-methoxy-phenyl, benzyl, methyl, phenyl, phenylmethyl, phenylethyl,4-chlorophenyl, 3,4-difluorophenyl, 3,4-dimethoxyphenyl,2-methoxyphenyl, 3-methoxyphenyl, 4-methoxyphenyl, and 2-nitrophenyl.19. The cell adhesion inhibitory compound according to claim 17, whereinR₄ is selected from the group consisting 4-methoxyphenyl,3,4-dimethoxyphenyl, 4-fluorophenyl, 4-carboxyphenyl,4-carbomethoxyphenyl, phenylethyl, phenylmethyl, allyl, and ethynyl. 20.The cell adhesion inhibitory compound according to claim 19, wherein R₄is selected from the group consisting of 4-methoxyphenyl,3,4-dimethoxyphenyl, 4-fluorophenyl, 4-carboxyphenyl,4-carbomethoxyphenyl, phenylethyl, and phenylmethyl.
 21. The celladhesion inhibitory compound according to claim 1, wherein Y is CO, orSO₂.
 22. The cell adhesion inhibitory compound according to claim 21,wherein Y is CO.
 23. The cell adhesion inhibitory compound according toclaim 1, wherein n is
 1. 24. The cell adhesion inhibitory compoundaccording to claim 2, selected from the group consisting of: β-Alanine,N-[[4-[[(phenylamino)carbonyl]amino]phenyl]acetyl]-L-methionyl-3-(4methoxyphenyl)-, (S)-, β-Alanine,N-[[4-[[(2-methylphenylamino)carbonyl]amino]phenyl]acetyl]-L-methionyl-3-(4methoxyphenyl)-,(S)-, β-Alanine,N-[[4-[[(2-methylphenylamino)carbonyl]amino]phenyl]acetyl]-L-leucinyl-3-(4fluorophenyl)-, (S)-, βAlanine,N6-(methanesulfonyl)-N2-[[4-[[[2-methylphenyl)amino]carbonyl]-amino]phenyl]acetyl]-L-lysyl-3-(4-carbomethoxyphenyl)-,(S)-, and β-Alanine,N-[[4-[[(2-methylphenylamino)carbonyl]amino]phenyl]acetyl]-L-methionyl-3-(4-(1-phenethyl)-,(S)-.
 25. The cell adhesion inhibitory compound according to claim 1,selected from the group consisting of: β-Alanine,N-[[4-[[(2-methylphenylamino)carbonyl]amino]phenyl]acetyl]-L-methionyl-3-(4-carbomethoxyphenyl)-,(S)-, β-Alanine,N-[[4-[[(phenylamino)carbonyl]amino]phenyl]acetyl]-L-methionyl-3-(4carbomethoxyphenyl)-,(S)-, β-Alanine,N-[[4-[[(2-methylphenylamino)carbonyl]amino]phenyl]acetyl]-L-leucyl-3-(ethynyl)-,(S)-, β-Alanine,N-[[4[[(2-methylphenylamino)carbonyl]amino]phenyl]acetyl]-L-leucyl-3-(allyl)-,(S)-, β-Alanine,N-[[4-[[(2-fluorophenylamino)carbonyl]amino]phenyl]acetyl]-L-leucyl-3-3,4-dimethoxyphenyl)-,(S)-, β-Alanine,N-[[4-[[(4-fluorophenylamino)carbonyl]amino]phenyl]acetyl]-L-leucyl-3-(3,4-dimethoxyphenyl)-(S)-, β-Alanine,N-[[4-[[(2-methylphenylamino)carbonyl]amino]phenyl]acetyl]-L-leucyl-3-(methyl)-,(S)-, β-Alanine,N-[[4-[[(2-methylphenylamino)carbonyl]amino]phenyl]acetyl[-L-leucyl-3-,(S)-, β-Alanine,N-[[4-[[(phenylamino)carbonyl]amino](3-methoxyphenyl)]acetyl]-L-leucyl-3-(3,4-dimethoxyphenyl)-,(S), and β-Alanine,N-[[4-[[(phenylamino)carbonyl]amino](3-methoxyphenyl)]acetyl]-L-methionyl-3-(3,4dimethoxyphenyl)-, (S).
 26. The cell adhesion inhibitory compoundaccording to claim 1, selected from the group consisting of: β-Alanine,N-[[4-[[(2-methylphenylamino)carbonyl]amino]phenyl]acetyl]-L-leucyl3(allyl),(S)-, β-Alanine,N-[[4-[[(phenylamino)carbonyl]amino](3-methoxyphenyl)]acetyl]-L-leucyl-3-(3,4-dimethoxyphenyl)-,(S), β-Alanine,N-[[4-[[(phenylamino)carbonyl]amino](3-methoxyphenyl)]acetyl]-L-methionyl-3-(3,4dimethoxyphenyl)-, (S).
 27. A pharmaceutical composition comprising acompound according to claim 1 in an amount effective for prevention,inhibition or suppression of VLA-4 mediated cell adhesion and apharmaceutically acceptable carrier.
 28. The pharmaceutical compositionaccording to claim 27, further comprising an agent selected from thegroup consisting of corticosteriods, bronchodilators, antiasthinatics,antiinflammatories, antirheumatics, inununosuppressants,antimetabolites, immunonodulators, antipsoriatics and antidiabetics. 29.A method of preventing, inhibiting or suppressing cell adhesion in amammal comprising the step of administering to said mammal thepharmaceutical composition according to claim
 27. 30. The methodaccording to claim 29, wherein said method is used for preventing,inhibiting or suppressing cell adhesion-associated inflammation.
 31. Themethod according to claim 30, wherein said method is used forpreventing, inhibiting or suppressing cell adhesion-associated immune orautoimmune response.
 32. The method according to claim 29, wherein saidmethod is used to treat or prevent a disease selected from the groupconsisting of asthma, arthritis, psoriasis, transplantation rejection,multiple sclerosis, diabetes and inflammatory bowel disease.
 33. A celladhesion inhibitory compound selected from a compound of the formula(I):

or a pharmaceutically acceptable derivative of (I), wherein: X is —CO₂H;Y is selected from the group consisting of —CO—, —SO₂— and —PO₂—; R₁ is(N′-Ar-urea)-substituted arylalkyl, wherein the urea is substituted atthe para position of an arylalkyl; R₂ is selected from the groupconsisting of hydrogen, aryl, alkyl, alkenyl, alkynyl, cycloalkyl,cycloalkenyl, and aryl-substituted alkyl; R₃ is selected from the groupconsisting of alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl,aralkyl, aryl-substituted alkenyl, aryl-substituted alkynyl,hydroxy-substituted alkyl, alkoxy-substituted alkyl,aralkoxy-substituted alkyl, amino-substituted alkyl, (aryl-substitutedalkyloxycarbonylamino)-substituted alkyl, thiol-substituted alkyl,alkylsulfonyl-substituted alkyl, (hydroxy-substitutedalkylthio)-substituted alkyl, thioalkoxy-substituted alkyl,acylamino-substituted alkyl, alkylsulfonylamino-substituted alkyl,arylsulfonylamino-substituted alkyl, N-(alkyl, alkenyl, or alkynyl)- orN,N-(dialkyl, dialkenyl, or dialkynyl)- or N,N-(alkyl,alkenyl)-aminocarbonyl-substituted alkyl, carboxyl-substituted alkyl,dialkylamino-substituted acylaminoalkyl and amino acid side chainsselected from the group consisting of arginine, asparagine, glutamine,S-methyl cysteine, methionine and corresponding sulfoxide and sulfonederivatives thereof, glycine, leucine, isoleucine, allo-isoleucine,tert-leucine, norleucine, phenylalanine, tyrosine, alanine, ornithine,glutamine, valine, threonine, serine, beta-cyanoalanine, andallothreonine; R₄ is selected from the group consisting of aryl, alkyl,cycloalkyl, alkenyl, cycloalkenyl, alkynyl, aryl-substituted alkyl,hydrogen, amido, mono- or dialkylaminocarbonyl, mono- ordiarylaminocarbonyl, alkylarylaminocarbonyl, diarylaminocarbonyl, mono-or diacylaminocarbonyl, aromatic acyl, alkyl optionally substituted bysubstituents selected from the group consisting of amino, carboxy,hydroxy, mercapto, mono- or dialkylamino, mono- or diarylamino,alkylarylamino, diarylammo, mono- or diacylamino, alkoxy, alkenoxy,aryloxy, thioalkoxy, thioalkenoxy, thioalkynoxy, and thioaryloxy; and nis 0, 1, or 2; provided that at least one of R₁-R₄ contains an arylgroup.
 34. The cell adhesion inhibitory compound according to claim 33,wherein R₁ is a (N′-Ar-urea), wherein the urea is substituted at thepara position of phenylmethyl.
 35. The cell adhesion inhibitory compoundaccording to claim 33, wherein R₁ is selected from the group consistingof 3-methoxy-4-(N-phenylurea)-phenylmethyl,4-(N′-phenylurea)-phenylmethyl,4-(N′-(2-methylphenyl)urea)-phenylmethyl, and3-methoxy-4-(N′-(2-methylphenyl)urea)-phenylmethyl.
 36. The celladhesion inhibitory compound according to claim 33, wherein R₂ ishydrogen, methyl, or phenacyl.
 37. The cell adhesion inhibitory compoundaccording to claim 33, wherein R₂ is hydrogen.
 38. The cell adhesioninhibitory compound according to claim 33, wherein R₃ is selected fromthe group consisting of 2-(methylsulfonyl)-ethyl,3-(hydroxy-propylthio)-methyl, 4-(methylsulfonylamino)-butyl,4-acetylaminobutyl, aminomethyl, benzyl, butyl, hydroxymethyl, isobutyl,methyl, methylthiomethyl, phenylmethyl, propyl,4-(benzloxycarbonylamino)-butyl, N,N-(methylpropargyl)amino,2-(methylthio)-ethyl, 2-(N,N-dimethylamino)ethyl, 4-amino-butyl,4-benzyloxyphenylmethyl, 2-benzylthiomethyl,t-butoxy-carbonylaminomethyl, sec-butyl, t-butyl,N,N-dimethyl-aminocarbonylmethyl, 1,1-ethano, 4-hydroxyphenylmethyl,1-hydroxyethyl, 1-methoxyethyl, 4-methoxyphenylmethyl, benzyloxymethyl,benzylthiomethyl, carbonylmethyl, 2-methylsulfinylethyl, 2-phenylethyl,asparagine side-chain, 4-(phenylurea)butyl, 4-(methylurea)butyl,4-methylsulfonylaminobutyl; hydroxymethylthiomethyl,2-methylsulfonylethyl, 4-propionylaminobutyl,4-ethoxycarbonylaminobutyl, methoxycarbonylaminobutyl,carbomethoxymethylthiomethyl, 4-t-butylureabutyl,carboxymethylthiomethyl, dimethylamidomethylthiomethyl,acetylaminopropyl; 3-methylureapropyl, 4-trifluoroacetylaminobutyl,dimethylaminomethylthiomethyl, dimethylaminoethylthiomethyl, and4-(dimethylaminoacetylamino)butyl.
 39. The cell adhesion inhibitorycompound according to claim 33, wherein R₃ is selected from the groupconsisting of 2-(methylsulfonyl)-ethyl, 3-(hydroxy-propylthio)-methyl,4-(methylsulfonylamino)-butyl, 4-acetylaminobutyl, aminomethyl, benzyl,butyl, hydroxymethyl, isobutyl, methyl, methylthiomethyl, phenylmethyl,propyl, 4-(benzyloxycarbonylamino)-butyl, N,N-(methylpropargyl)amino,2-(methylthio)-ethyl, 2-(N,N-dimethylamino)ethyl, 4-amino-butyl,4-benzyloxyphenylmethyl, 2-benzylthiomethyl,t-butoxy-carbonylaminomethyl, sec-butyl, t-butyl,N,N-dimethyl-aminocarbonylmethyl, 1,1-ethano, 4-hydroxyphenylmethyl,1-hydroxyethyl, 1-methoxyethyl, 4-methoxyphenylmethyl, benzloxymethyl,benzylthiomethyl, carbonylmethyl, 2-methylsulfinylethyl, 2-phenylethyl,and asparagine side-chain.
 40. The cell adhesion inhibitory compoundaccording to claim 33, wherein R₃ is selected from the group consistingof isobutyl, 2-(methylthio)-ethyl, 3-(hydroxypropylthio)-methyl,2-(methylsulfonyl)-ethyl, 4-acetylamino-butyl,4-(methylsulfonylamino)-butyl, and 4-(ethoxycarbonylamino)butyl.
 41. Thecell adhesion inhibitory compound according to claim 33, wherein R₃ isselected from the group consisting of isobutyl, 2-(methylthio)-ethyl,3-(hydroxypropylthio)-methyl, 2-(methylsulfonyl)-ethyl,4-acetylamino-butyl, and 4-(methylsulfonylamino)-butyl.
 42. The celladhesion inhibitory compound according to claim 33, wherein R₄ isselected from the group consisting of 4-carbomethoxy, 4-fluorophenyl,4-methoxyphenyl, benzyl, methyl, phenyl, phenylmethyl, phenylethyl,4-chlorophenyl, 3,4-difluorophenyl, 3,4-dimethoxyphenyl,2-methoxyphenyl, 3-methoxyphenyl, 4-methoxyphenyl, 2-nitrophenyl,4-phenoxyphenyl, 4-ethoxyphenyl, 4-nitrophenyl, 4-acetylaminophenyl,4-methylureaphenyl, 2-fluorophenyl, naphthyl, 3-fluorophenyl,3-nitrophenyl, hydrogen, 2-nitrophenyl, 4-cyanophenyl, 3-methoxyphenyl,4-methylsulfonylamino, 3-cyanophenyl, 4-propionylamino, 4-aminophenyl,3-aminophenyl, 4-trifluoromethoxyphenyl, 4-methylphenyl,4-amino-3-nitrophenyl, 4-hydroxy-3-methoxyphenyl, 4-hexyloxyphenyl,4-methylthiophenyl, 4-dimethylaminophenyl, 3-hydroxy-4-nitrophenyl,n-pentyl, carboxymethyl, 2-carboxyethyl, ethynyl, 2-propenyl,2-propynyl, methyl, and propyl.
 43. The cell adhesion inhibitorycompound according to claim 33, wherein R₄ is selected from the groupconsisting of 4-carbomethoxyphenyl, 4-carboxyphenyl, 4-fluorophenyl,4-methoxyphenyl, benzyl, methyl, phenyl, phenylmethyl, phenylethyl,4-chlorophenyl, 3,4-difluorophenyl, 3,4-dimethoxyphenyl,2-methoxyphenyl, 3-methoxyphenyl, 4-methoxyphenyl, and 2-nitrophenyl.44. The cell adhesion inhibitory compound according to claim 33, whereinR₄ is selected from the group consisting 4-methoxyphenyl,3,4-dimethoxyphenyl, 4-fluorophenyl, 4-carboxyphenyl,4-carbomethoxyphenyl, phenylethyl, phenylmethyl, allyl, ethynyl, and3,4-methylenedioxyphenyl.
 45. The cell adhesion inhibitory compoundaccording to claim 33, wherein R₄ is selected from the group consisting4-methoxyphenyl, 3,4-dimethoxyphenyl, 4-fluorophenyl, 4-carboxyphenyl,4-carbomethoxyphenyl, phenylethyl, and phenylmethyl.
 46. The celladhesion inhibitory compound of claim 1, wherein Y is CO, R₁ is(N-Ar′-urea)-para-substituted aralkyl group; R₂ is H; R₃ is selectedfrom the group consisting of isobutyl, 2-(methylthio)-ethyl,3-(hydroxypropylthio)-methyl, 2-(methylsulfonyl)-ethyl,4-acetylamino-butyl, 4-(methylsulfonylamino)-butyl, and4-(ethoxycarbonylamino)butyl; and R₄ is selected from the groupconsisting of 4-carbomethoxyphenyl, 4-carboxyphenyl, 4-fluorophenyl,4-methoxyphenyl, benzyl, methyl, phenyl, phenylmethyl, phenylethyl,4-chlorophenyl, 3,4-difluorophenyl, 3,4-dimethoxyphenyl,2-methoxyphenyl, 3-methoxyphenyl, 4-methoxyphenyl, and 2-nitrophenyl.47. The cell adhesion inhibitory compound of claim 1, wherein Y is SO₂;R₂ is H; R₃ is selected from the group consisting of isobutyl,2-(methylthio)-ethyl, 3-(hydroxypropylthio)-methyl,2-(methylsulfonyl)-ethyl, 4-acetylamino-butyl,4-(methylsulfonylamino)-butyl, and 4-(ethoxycarbonylamino)butyl; and R₄is selected from the group consisting of 4-carbomethoxy-phenyl,4-carboxyphenyl, 4-fluorophenyl, 4-methoxyphenyl, benzyl, methyl,phenyl, phenylmethyl, phenylethyl, 4-chlorophenyl, 3,4-difluorophenyl,3,4-dimethoxyphenyl, 2-methoxyphenyl, 3-methoxyphenyl, 4-methoxyphenyl,and 2-nitrophenyl.
 48. The cell adhesion inhibitory compound of claim47, wherein R₁ is (N-Ar′-urea)-para-substituted aralkyl group.
 49. Thecell adhesion inhibitory compound of claim 1, wherein R₂ is selectedfrom the group consisting of aryl, alkyl, alkenyl, alkynyl, cycloalkyl,cycloalkenyl, and aryl-substituted alkyl.
 50. The cell adhesioninhibitory compound of claim 1, wherein R₁ is selected from the groupconsisting of alkenyl, alkynyl, cycloalkyl, aryl-fused cycloalkyl,cycloalkenyl, aryl, aralkyl, aryl-substituted alkenyl or alkynyl,cycloalkenyl-substituted alkyl, alkylamino, alkenylamino oralkynylamino, aryl-substituted alkylamino, aryl-substituted alkenylaminoor alkynylamino, N-alkylurea-substituted alkyl, N-arylurea-substitutedalkyl, and aminocarbonyl-substituted alkyl.
 51. The cell adhesioninhibitory compound of claim 1, wherein Y is CO, R₁ is(N-Ar′-urea)-para-substituted aralkyl group; R₂ is H; R₃ is selectedfrom the group consisting of isobutyl, 2-(methylthio)-ethyl,3-(hydroxypropylthio)-methyl, 2-(methylsulfonyl)-ethyl,4-acetylamino-butyl, 4-(methylsulfonylamino)-butyl, and4-(ethoxycarbonylamino)butyl; and R₄ is selected from the groupconsisting of 4 carbomethoxy-phenyl, 4-carboxyphenyl, 4-fluorophenyl,4-methoxy-phenyl, benzyl, methyl, phenyl, phenylmethyl, phenylethyl,4-chlorophenyl, 3,4-difluorophenyl, 3,4-dimethoxyphenyl,2-methoxyphenyl, 3-methoxyphenyl, 4-methoxyphenyl, and 2-nitrophenyl.52. The cell adhesion inhibitory compound of claim 1, wherein Y is SO₂;R₂ is H; R₃ is selected from the group consisting of isobutyl,2-(methylthio)-ethyl, 3-(hydroxypropylthio)-methyl,2-(methylsulfonyl)-ethyl, 4-acetylamino-butyl,4-(methylsulfonylamino)-butyl, and 4-(ethoxycarbonylamino)butyl; and R₄is selected from the group consisting of 4-carbomethoxy-phenyl,4-carboxyphenyl, 4-fluorophenyl, 4-methoxyphenyl, benzyl, methyl,phenyl, phenylmethyl, phenylethyl, 4-chlorophenyl, 3,4-difluorophenyl,3,4-dimethoxyphenyl, 2-methoxyphenyl, 3-methoxyphenyl, 4-methoxyphenyl,and 2-nitrophenyl.
 53. The cell adhesion inhibitory compound of claim52, wherein R₁ is (N-Ar′-urea)-para-substituted aralkyl group.